Bioavailable Diacylhydrazine Ligands for Modulations the Expression of Exogenous Genes via an Ecdysone Receptor Complex

ABSTRACT

The present invention relates to non-steroidal ligands for use in nuclear receptor-based inducible gene expression system, and a method to modulate exogenous gene expression in which an ecdysone receptor complex comprising: a DNA binding domain; a ligand binding domain; a transactivation domain; and a ligand is contacted with a DNA construct comprising: the exogenous gene and a response element; wherein the exogenous gene is under the control of the response element and binding of the DNA binding domain to the response element in the presence of the ligand results in activation or suppression of the gene.

This application claims priority to U.S. provisional application No.60/455,741 filed Feb. 28, 2003.

FIELD OF THE INVENTION

This invention relates to the field of biotechnology or geneticengineering. Specifically, this invention relates to the field of geneexpression. More specifically, this invention relates to non-steroidalligands for natural and mutated nuclear receptors and their use in anuclear receptor-based inducible gene expression system and methods ofmodulating the expression of a gene within a host cell using theseligands and inducible gene expression system.

BACKGROUND OF THE INVENTION

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties. However, the citation ofany reference herein should not be construed as an admission that suchreference is available as “Prior Art” to the instant application.

In the field of genetic engineering, precise control of gene expressionis a valuable tool for studying, manipulating, and controllingdevelopment and other physiological processes. Gene expression is acomplex biological process involving a number of specificprotein-protein interactions. In order for gene expression to betriggered, such that it produces the RNA necessary as the first step inprotein synthesis, a transcriptional activator must be brought intoproximity of a promoter that controls gene transcription. Typically, thetranscriptional activator itself is associated with a protein that hasat least one DNA binding domain that binds to DNA binding sites presentin the promoter regions of genes. Thus, for gene expression to occur, aprotein comprising a DNA binding domain and a transactivation domainlocated at an appropriate distance from the DNA binding domain must bebrought into the correct position in the promoter region of the gene.

The traditional transgenic approach utilizes a cell-type specificpromoter to drive the expression of a designed transgene. A DNAconstruct containing the transgene is first incorporated into a hostgenome. When triggered by a transcriptional activator, expression of thetransgene occurs in a given cell type.

Another means to regulate expression of foreign genes in cells isthrough inducible promoters. Examples of the use of such induciblepromoters include the PR1-a promoter, prokaryotic repressor-operatorsystems, immunosuppressive-immunophilin systems, and higher eukaryotictranscription activation systems such as steroid hormone receptorsystems and are described below.

The PR1-a promoter from tobacco is induced during the systemic acquiredresistance response following pathogen attack. The use of PR1-a may belimited because it often responds to endogenous materials and externalfactors such as pathogens, UV-B radiation, and pollutants. Generegulation systems based on promoters induced by heat shock, interferonand heavy metals have been described (Wurn et al., 1986, Proc. Natl.Acad. Sci. USA 83:5414-5418; Arnheiter et al., 1990 Cell 62:51-61;Filmus et al., 1992 Nucleic Acids Research 20:27550-27560). However,these systems have limitations due to their effect on expression ofnon-target genes. These systems are also leaky.

Prokaryotic repressor-operator systems utilize bacterial repressorproteins and the unique operator DNA sequences to which they bind. Boththe tetracycline (“Tet”) and lactose (“Lac”) repressor-operator systemsfrom the bacterium Escherichia coli have been used in plants and animalsto control gene expression. In the Tet system, tetracycline binds to theTetR repressor protein, resulting in a conformational change thatreleases the repressor protein from the operator which as a resultallows transcription to occur. In the Lac system, a lac operon isactivated in response to the presence of lactose, or synthetic analogssuch as isopropyl-b-D-thiogalactoside. Unfortunately, the use of suchsystems is restricted by unstable chemistry of the ligands, i.e.tetracycline and lactose, their toxicity, their natural presence, or therelatively high levels required for induction or repression. For similarreasons, utility of such systems in animals is limited.

Immunosuppressive molecules such as FK506, rapamycin and cyclosporine Acan bind to immunophilins FKBP12, cyclophilin, etc. Using thisinformation, a general strategy has been devised to bring together anytwo proteins simply by placing FK506 on each of the two proteins or byplacing FK506 on one and cyclosporine A on another one. A synthetichomodimer of FK506 (FK1012) or a compound resulted from fusion ofFK506-cyclosporine (FKCsA) can then be used to induce dimerization ofthese molecules (Spencer et al., 1993, Science 262:1019-24; Belshaw etal., 1996 Proc Natl Acad Sci USA 93:4604-7). Gal4 DNA binding domainfused to FKBP12 and VP16 activator domain fused to cyclophilin, andFKCsA compound were used to show heterodimerization and activation of areporter gene under the control of a promoter containing Gal4 bindingsites. Unfortunately, this system includes immunosuppressants that canhave unwanted side effects and therefore, limits its use for variousmammalian gene switch applications.

Higher eukaryotic transcription activation systems such as steroidhormone receptor systems have also been employed. Steroid hormonereceptors are members of the nuclear receptor superfamily and are foundin vertebrate and invertebrate cells. Unfortunately, use of steroidalcompounds that activate the receptors for the regulation of geneexpression, particularly in plants and mammals, is limited due to theirinvolvement in many other natural biological pathways in such organisms.In order to overcome such difficulties, an alternative system has beendeveloped using insect ecdysone receptors (EcR).

Growth, molting, and development in insects are regulated by theecdysone steroid hormone (molting hormone) and the juvenile hormones(Dhadialla, et al., 1998. Annu. Rev. Entomol. 43: 545-569). Themolecular target for ecdysone in insects consists of at least ecdysonereceptor (EcR) and ultraspiracle protein (USP). EcR is a member of thenuclear steroid receptor super family that is characterized by signatureDNA and ligand binding domains, and an activation domain (Koelle et al.1991, Cell, 67:59-77). EcR receptors are responsive to a number ofsteroidal compounds such as ponasterone A and muristerone A. Recently,non-steroidal compounds with ecdysteroid agonist activity have beendescribed, including the commercially available insecticidestebufenozide and methoxyfenozide that are marketed world wide by Rohmand Haas Company (see International Patent Application No.PCT/EP96/00686 and U.S. Pat. No. 5,530,028). Both analogs haveexceptional safety profiles to other organisms.

The insect ecdysone receptor (EcR) heterodimerizes with Ultraspiracle(USP), the insect homologue of the mammalian RXR, and binds ecdysteroidsand ecdysone receptor response elements and activate transcription ofecdysone responsive genes. The EcR/USP/ligand complexes play importantroles during insect development and reproduction. The EcR is a member ofthe steroid hormone receptor superfamily and has five modular domains,A/B (transactivation), C (DNA binding, heterodimerization)), D (Hinge,heterodimerization), E (ligand binding, heterodimerization andtransactivation and F (transactivation) domains. Some of these domainssuch as A/B, C and E retain their function when they are fused to otherproteins.

Tightly regulated inducible gene expression systems or “gene switches”are useful for various applications such as gene therapy, large scaleproduction of proteins in cells, cell based high throughput screeningassays, functional genomics and regulation of traits in transgenicplants and animals.

The first version of EcR-based gene switch used Drosophila melanogasterEcR (DmEcR) and Mus musculus RXR (MmRXR) and showed that these receptorsin the presence of steroid, ponasteroneA, transactivate reporter genesin mammalian cell lines and transgenic mice (Christopherson K. S., MarkM. R., Baja J. V., Godowski P. J. 1992, Proc. Natl. Acad. Sci. U.S.A.89: 6314-6318; No D., Yao T. P., Evans R. M., 1996, Proc. Natl. Acad.Sci. U.S.A. 93: 3346-3351). Later, Suhr et al. 1998, Proc. Natl. Acad.Sci. 95:7999-8004 showed that non-steroidal ecdysone agonist,tebufenozide, induced high level of transactivation of reporter genes inmammalian cells through Bombyx mori EcR (BmEcR) in the absence ofexogenous heterodimer partner.

International Patent Applications No. PCT/US97/05330 (WO 97/38117) andPCT/US99/08381 (WO99/58155) disclose methods for modulating theexpression of an exogenous gene in which a DNA construct comprising theexogenous gene and an ecdysone response element is activated by a secondDNA construct comprising an ecdysone receptor that, in the presence of aligand therefor, and optionally in the presence of a receptor capable ofacting as a silent partner, binds to the ecdysone response element toinduce gene expression. The ecdysone receptor of choice was isolatedfrom Drosophila melanogaster. Typically, such systems require thepresence of the silent partner, preferably retinoid X receptor (RXR), inorder to provide optimum activation. In mammalian cells, insect ecdysonereceptor (EcR) heterodimerizes with retinoid X receptor (RXR) andregulates expression of target genes in a ligand dependent manner.International Patent Application No. PCT/US98/14215 (WO 99/02683)discloses that the ecdysone receptor isolated from the silk moth Bombyxmori is functional in mammalian systems without the need for anexogenous dimer partner.

U.S. Pat. No. 6,265,173 B1 discloses that various members of thesteroid/thyroid superfamily of receptors can combine with Drosophilamelanogaster ultraspiracle receptor (USP) or fragments thereofcomprising at least the dimerization domain of USP for use in a geneexpression system. U.S. Pat. No. 5,880,333 discloses a Drosophilamelanogaster EcR and ultraspiracle (USP) heterodimer system used inplants in which the transactivation domain and the DNA binding domainare positioned on two different hybrid proteins. Unfortunately, theseUSP-based systems are constitutive in animal cells and therefore, arenot effective for regulating reporter gene expression.

In each of these cases, the transactivation domain and the DNA bindingdomain (either as native EcR as in International Patent Application No.PCT/US98/14215 or as modified EcR as in International Patent ApplicationNo. PCT/US97/05330) were incorporated into a single molecule and theother heterodimeric partners, either USP or RXR, were used in theirnative state.

Drawbacks of the above described EcR-based gene regulation systemsinclude a considerable background activity in the absence of ligands andnon-applicability of these systems for use in both plants and animals(see U.S. Pat. No. 5,880,333). Therefore, a need exists in the art forimproved EcR-based systems to precisely modulate the expression ofexogenous genes in both plants and animals. Such improved systems wouldbe useful for applications such as gene therapy, large-scale productionof proteins and antibodies, cell-based high throughput screening assays,functional genomics and regulation of traits in transgenic animals. Forcertain applications such as gene therapy, it may be desirable to havean inducible gene expression system that responds well to syntheticnon-steroid ligands and at the same is insensitive to the naturalsteroids. Thus, improved systems that are simple, compact, and dependenton ligands that are relatively inexpensive, readily available, and oflow toxicity to the host would prove useful for regulating biologicalsystems.

Recently, it has been shown that an ecdysone receptor-based induciblegene expression system in which the transactivation and DNA bindingdomains are separated from each other by placing them on two differentproteins results in greatly reduced background activity in the absenceof a ligand and significantly increased activity over background in thepresence of a ligand (pending application PCT/US01/09050, incorporatedherein in its entirety by reference). This two-hybrid system is asignificantly improved inducible gene expression modulation systemcompared to the two systems disclosed in applications PCT/US97/05330 andPCT/US98/14215. The two-hybrid system exploits the ability of a pair ofinteracting proteins to bring the transcription activation domain into amore favorable position relative to the DNA binding domain such thatwhen the DNA binding domain binds to the DNA binding site on the gene,the transactivation domain more effectively activates the promoter (see,for example, U.S. Pat. No. 5,283,173). Briefly, the two-hybrid geneexpression system comprises two gene expression cassettes; the firstencoding a DNA binding domain fused to a nuclear receptor polypeptide,and the second encoding a transactivation domain fused to a differentnuclear receptor polypeptide. In the presence of ligand, the interactionof the first polypeptide with the second polypeptide effectively tethersthe DNA binding domain to the transactivation domain. Since the DNAbinding and transactivation domains reside on two different molecules,the background activity in the absence of ligand is greatly reduced.

A two-hybrid system also provides improved sensitivity to non-steroidalligands for example, diacylhydrazines, when compared to steroidalligands for example, ponasterone A (“PonA”) or muristerone A (“MurA”).That is, when compared to steroids, the non-steroidal ligands providehigher activity at a lower concentration. In addition, sincetransactivation based on EcR gene switches is often cell-line dependent,it is easier to tailor switching systems to obtain maximumtransactivation capability for each application. Furthermore, thetwo-hybrid system avoids some side effects due to overexpression of RXRthat often occur when unmodified RXR is used as a switching partner. Ina preferred two-hybrid system, native DNA binding and transactivationdomains of EcR or RXR are eliminated and as a result, these hybridmolecules have less chance of interacting with other steroid hormonereceptors present in the cell resulting in reduced side effects.

With the improvement in ecdysone receptor-based gene regulation systemsthere is an increase in their use in various applications resulting inincreased demand for ligands with higher activity than those currentlyexist. U.S. Pat. No. 6,258,603 B1 (and patents cited therein) discloseddibenzoylhydrazine ligands, however, a need exists for additionalligands with different structures and physicochemical properties. Wehave discovered novel diacylhydrazine ligands which have not previouslybeen described or shown to have the ability to modulate the expressionof transgenes.

SUMMARY OF THE INVENTION

The present invention relates to non-steroidal ligands for use innuclear receptor-based inducible gene expression system, and methods ofmodulating the expression of a gene within a host cell using theseligands with nuclear receptor-based inducible gene expression systems.

Applicants' invention also relates to methods of modulating geneexpression in a host cell using a gene expression modulation system witha ligand of the present invention. Specifically, Applicants' inventionprovides a method of modulating the expression of a gene in a host cellcomprising the steps of: a) introducing into the host cell a geneexpression modulation system according to the invention; b) introducinginto the host cell a gene expression cassette comprising i) a responseelement comprising a domain to which the DNA binding domain from thefirst hybrid polypeptide of the gene expression modulation system binds;ii) a promoter that is activated by the transactivation domain of thesecond hybrid polypeptide of the gene expression modulation system; andiii) a gene whose expression is to be modulated; and c) introducing intothe host cell a ligand; whereby upon introduction of the ligand into thehost cell, expression of the gene is modulated. Applicants' inventionalso provides a method of modulating the expression of a gene in a hostcell comprising a gene expression cassette comprising a response elementcomprising a domain to which the DNA binding domain from the firsthybrid polypeptide of the gene expression modulation system binds; apromoter that is activated by the transactivation domain of the secondhybrid polypeptide of the gene expression modulation system; and a genewhose expression is to be modulated; wherein the method comprises thesteps of: a) introducing into the host cell a gene expression modulationsystem according to the invention; and b) introducing into the host cella ligand; whereby upon introduction of the ligand into the host,expression of the gene is modulated.

FIG. 1. Schematic of switch construct CVBE, and the reporter construct6XEcRE Lac Z. Flanking both constructs are long terminal repeats, G418and puromycin are selectable markers, CMV is the cytomegaloviruspromoter, VBE is coding sequence for amino acids 26-546 from Bombyx moriEcR inserted downstream of the VP16 transactivation domain, 6XEcRE issix copies of the ecdysone response element, lacZ encodes for thereporter enzyme β-galactosidase.

DETAILED DESCRIPTION OF THE INVENTION

Applicants' invention provides ligands for use with ecdysonereceptor-based inducible gene expression system useful for modulatingexpression of a gene of interest in a host cell. In a particularlydesirable embodiment, Applicants' invention provides an inducible geneexpression system that has a reduced level of background gene expressionand responds to submicromolar concentrations of non-steroidal ligand.Thus, Applicants' ligands and inducible gene expression system and itsuse in methods of modulating gene expression in a host cell overcome thelimitations of currently available inducible expression systems andprovide the skilled artisan with an effective means to control geneexpression.

The present invention is useful for applications such as gene therapy,large scale production of proteins and antibodies, cell-based highthroughput screening assays, functional genomics, proteomics,metabolomics, and regulation of traits in transgenic organisms, wherecontrol of gene expression levels is desirable. An advantage ofApplicants' invention is that it provides a means to regulate geneexpression and to tailor expression levels to suit the user'srequirements.

The present invention pertains to compounds of the general formula:

wherein X and X′ are independently O or S;

A is unsubstituted or substituted phenyl wherein the substituents areindependently 1 to 5H; halo; nitro; cyano; hydroxy; amino(—NR^(a)R^(b)); alkylaminolkyl (—(CH₂)_(n)NR^(a)R^(b)); (C₁-C₆)alkyl;(C₁-C₆)haloalkyl; (C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy;phenoxy; (C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;(C₁-C₆)alkenyloxy(C₁-C₆)alkyl; (C₁-C₆)alkoxy(C₁-C₆)alkoxy;(C₁-C₆)alkanoyloxy(C₁-C₆)alkyl; (C₂-C₆)alkenyl optionally substitutedwith halo, cyano, (C₁-C₄)alkyl, or (C₁-C₄)alkoxy; (C₂-C₆)alkynyloptionally substituted with halo or (C₁-C₄)alkyl; formyl; carboxy;(C₁-C₆)alkylcarbonyl; (C₁-C₆)haloalkylcarbonyl; benzoyl;(C₁-C₆)alkoxycarbonyl; (C₁-C₆)haloalkoxycarbonyl; (C₁-C₆)alkanoyloxy(—OCOR^(a)); carboxamido (—CONR^(a)R^(b)); amido(—NR^(a)COR^(b));alkoxycarbonylamino (—NR^(a)CO₂R^(b)); alkylaminocarbonylamino(—NR^(a)CONR^(b)R^(c)); mercapto; (C₁-C₆)alkylthio; (C₁-C₆)alkylsulfonyl; (C₁-C₆)alkylsulfonyl(C₁-C₆)alkyl; (C₁-C₆)alkylsulfoxido(—S(O)R^(a)); (C₁-C₆)alkylsulfoxido(C₁-C₆)alkyl —(CH₂)_(n)S(O)R^(a));sulfamido (—SO₂NR^(a)R^(b)); —SO₃H; or unsubstituted or substitutedphenyl wherein the substituents are independently 1 to 3 halo, nitro,(C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or when one or both of twoadjacent positions on the phenyl ring are substituted, the attachedatoms may form the phenyl-connecting termini of a linkage selected fromthe group consisting of (—OCH₂O—), (—OCH(CH₃)O—), (—OCH₂CH₂O—),(—OCH(CH₃)CH₂O—), (—S—CH═N—), (—CH₂OCH₂O—), (—O(CH₂)₃—), (═N—O—N═),(—C═CH—NH—), (—OCF₂O—), (—N—CH═N—), (—CH₂CH₂O—), and (—(CH₂)₄ ⁻);

B is

-   -   (a) unsubstituted or substituted phenyl wherein the substituents        are independently 1 to 5H; halo; nitro; cyano; hydroxy; amino        (—NR^(a)R^(b)); alkylaminolkyl (—(CH₂)_(n)NR^(a)R^(b));        (C₁-C₆)alkyl; (C₁-C₆)haloalkyl; (C₁-C₆)cyanoalkyl;        (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy; phenoxy; (C₁-C₆)haloalkoxy;        (C₁-C₆)alkoxy(C₁-C₆)alkyl; (C₁-C₆)alkenyloxy(C₁-C₆)alkyl;        (C₁-C₆)alkoxy(C₁-C₆)alkoxy; (C₁-C₆)alkanoyloxy(C₁-C₆)alkyl;        (C₂-C₆)alkenyl optionally substituted with halo, cyano,        (C₁-C₄)alkyl, or (C₁-C₄)alkoxy; (C₂-C₆)alkynyl optionally        substituted with halo or (C₁-C₄)alkyl; formyl; carboxy;        (C₁-C₆)alkylcarbonyl; (C₁-C₆)haloalkylcarbonyl; benzoyl;        (C₁-C₆)alkoxycarbonyl; (C₁-C₆)haloalkoxycarbonyl;        (C₁-C₆)alkanoyloxy (—OCOR^(a)); carboxamido (—CONR^(a)R^(b));        amido (—NR^(a)COR^(b)); alkoxycarbonylamino (—NR^(a)CO₂R^(b));        alkylaminocarbonylamino (—NR^(a)CONR^(b)R^(c)); mercapto;        (C₁-C₆)alkylthio; (C₁-C₆)alkylsulfonyl;        (C₁-C₆)alkylsulfonyl(C₁-C₆)alkyl; (C₁-C₆)alkylsulfoxido        (—S(O)R^(a)); (C₁-C₆)alkylsulfoxido(C₁-C₆)alkyl        (—CH₂)_(n)S(O)R^(a)); sulfamido (—SO₂NR^(a)R^(b)); —SO₃H; or        unsubstituted or substituted phenyl wherein the substituents are        independently 1 to 3 halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl,        or amino; or when one or both of two adjacent positions on the        phenyl ring are substituted, the attached atoms may form the        phenyl-connecting termini of a linkage selected from the group        consisting of (—OCH₂O—), (—OCH(CH₃)O—), (—OCH₂CH₂O—),        (—OCH(CH₃)CH₂O—), (—S—CH═N—), (—CH₂OCH₂O—), (—O(CH₂)₃—),        (═N—O—N═), (—C═CH—NH—), (—OCF₂O—), (—NH—CH═N—), (—CH₂CH₂O—), and        (—(CH₂)₄—);    -   (b) unsubstituted 6-membered heterocycle or substituted        6-membered heterocycle having 1-3 nitrogen atoms and 3-5 nuclear        carbon atoms where the substituents are from one to three of the        same or different halo; nitro; hydroxy; (C₁-C₆)alkyl;        (C₁-C₆)alkoxy; (C₁-C₆)thioalkoxy; carboxy;        (C₁-C₆)alkoxycarbonyl; (C₁-C₆)carboxyalkyl;        (C₁-C₆)alkoxycarbonylalkyl having independently the stated        number of carbon atoms in each alkyl group; —CONR^(a)R^(b);        amino; (C₁-C₆)alkylamino; (C₁-C₆)dialkylamino having        independently the stated number of carbon atoms in each alkyl        group; haloalkyl including —CF₃; —C═N—NHC(O)NR^(a)R^(b); or        —C═N—NHC(O)C(O)NR^(a)R^(b); or    -   (c) 5-benzimidazolyl; 1-trityl-5-benzimidazolyl;        3-trityl-5-benzimidazolyl; 1H-indazole-3-yl;        1-trityl-1H-indazole-3-yl; or 1-(C₁-C₆)alkyl-1H-indole-2-yl;

E is unsubstituted or substituted (C₄-C₁₀) branched alkyl wherein thesubstituents are independently 1-4 cyano; halo; (C₅-C₆)cycloalkyl;phenyl; (C₂-C₃)alkenyl; hydroxy, (C₁-C₆)alkoxy; carboxy;(C₁-C₆)alkoxycarbonyl; (C₁-C₆)alkanoyloxy (—OCOR^(a)); formyl;(C₁-C₆)trialkylsilyloxy having independently the stated number of carbonatoms in each alkyl group; —C═N—OR^(a); —C═N—R^(d);—C═N—NHC(O)NR^(a)R^(b); or —C═N—NHC(O)C(O)NR^(a)R^(b);

wherein R^(a), R^(b), and R^(c) are independently H, (C₁-C₆)alkyl, orphenyl; R^(d) is hydroxy(C₁-C₆)alkyl; and n=1-4; and

G is H or CN;

provided that:

1) when E is unsubstituted or substituted (C₄-C₁₀) branched alkylwherein the substituents are independently 1-4 cyano; halo;(C₂-C₃)alkenyl; carboxy; or (C₁-C₆)alkoxycarbonyl;

then B is

-   -   (a) substituted phenyl which bears at least one        —C═N—NHC(O)NR^(a)R^(b) or —C═N—NHC(O)C(O)NR^(a)R^(b) group;    -   (b) substituted 6-membered heterocycle having 1-3 nitrogen atoms        and 3-5 nuclear carbon atoms which bears at least one haloalkyl        group; or    -   (c) 5-benzimidazolyl; 1-trityl-5-benzimidazolyl;        3-trityl-5-benzimidazolyl; 1H-indazole-3-yl;        1-trityl-1H-indazole-3-yl; or 1-(C₁-C₆)alkyl-1H-indole-2-yl;

wherein R^(a), R^(b) are independently H, (C₁-C₆)alkyl, or phenyl; or

2) when E is a substituted (C₄-C₁₀) branched alkyl which bears at leastone of phenyl; hydroxy, (C₁-C₆)alkoxy; or formyl;

then B is

-   -   (a) substituted phenyl which bears at least one        —C═N—NHC(O)NR^(a)R^(b) or —C═N—NHC(O)C(O)NR^(a)R^(b) group;    -   (b) substituted or unsubstituted 6-membered heterocycle having        1-3 nitrogen atoms and 3-5 nuclear carbon atoms; or    -   (c) 5-benzimidazolyl; 1-trityl-5-benzimidazolyl;        3-trityl-5-benzimidazolyl; 1H-indazole-3-yl;        1-trityl-1H-indazole-3-yl; or 1-(C₁-C₆)alkyl-1H-indole-2-yl;

wherein R^(a) and R^(b) are independently H, (C₁-C₆)alkyl, or phenyl.

Compounds of the general formula are preferred when X and X′ are O and Gis H.

Compounds of the present invention most preferred are the following:

Compound A B E RG-100864 4-Cl—Ph Ph t-Bu RG-101013 4-Et—Ph 2-NO₂—Ph t-BuRG-101542 4-CH₃—Ph 3-CH₃, 5-CH₃—Ph t-Bu RG-102125 4-Et—Ph 3-CH₃,5-CH₃—Ph t-Bu RG-100801 2,6-di-F—Ph 3-Cl, 5-Cl—Ph t-Bu RG-101202 2-CH₃,3-Cl—Ph 3-Cl—Ph t-Bu RG-101248 2-Cl, 3-OMe—Ph 2-Cl-5-CH₃—Ph t-BuRG-101664 2-CH₃, 3-Cl—Ph 3-CH₃-4-Br—Ph t-Bu RG-101862 4-Et—Ph3,5-di-CH₃-4-Cl—Ph t-Bu RG-101863 4-Et—Ph 3,4-di-CH₃-5-Cl—Ph t-BuRG-101057 4-OCH₃—Ph 2-Cl-4-F—Ph t-Bu RG-101774 4-Et—Ph 3-CH₃, 5-Cl—Pht-Bu RG-102592 4-Et—Ph 2-Et—Ph t-Bu RG-101376 4-OCH₃—Ph 3-Cl, 5-Cl—Pht-Bu RG-101398 4-Et—Ph 2-NO₂-5-CH₃—Ph t-Bu RG-100875 4-CH₂CN—Ph 3-CH₃,5-CH₃—Ph t-Bu RG-100694 2-CH₃, 3-OMe—Ph 3-CH₃—Ph t-Bu RG-101759 4-Br—Ph3-Cl, 5-Cl—Ph t-Bu RG-100915 2-CH₃, 3-NO₂—Ph 3-CH₃, 5-CH₃—Ph t-BuRG-100763 2-CH₃, 3-CH₃—Ph 2,5-di-OCH₃—Ph t-Bu RG-101178 2-CH₃, 3-CH₃—Ph2-OCH₃-5-Cl—Ph t-Bu RG-100568 2-NO₂, 3-OMe—Ph 3-CH₃, 5-CH₃—Ph t-BuRG-100764 2-CH₃, 3-CH₃—Ph 3-OMe, 5-OMe—Ph t-Bu RG-101864 3-Cl, 4-Et—Ph3-CH₃, 5-CH₃—Ph t-Bu RG-100342 4-CH(OH)CH₃—Ph 3-F, 5-F—Ph t-Bu RG-1013162-CH₃, 3-NMe₂—Ph 3-Cl, 5-Cl—Ph t-Bu RG-100814 2-CH₃, 3-Ac—Ph 3-CH₃,5-CH₃—Ph t-Bu RG-100749 2-CH₃, 3-OAc—Ph 3-CH₃, 5-CH₃—Ph t-Bu RG-1017342-CH₃, 3-I—Ph 3-CH3, 5-CH₃—Ph t-Bu RG-101408 2-CH₃, 3-OMe—Ph 3-Cl,5-Br—Ph t-Bu RG-101670 2-CH₃, 3-Oi—Pr—Ph 3-CH₃, 5-CH₃—Ph t-Bu RG-1001272-CH₃, 3-OCH3—Ph 2-Cl-3-pyridyl t-Bu RG-100766 2-CH₃, 3-OMe—Ph2-OCH₃-5-CH₃—Ph t-Bu RG-100603 2-CH₃, 3-OMe—Ph 2,5-F—Ph t-Bu RG-1010622-CH₃, 3-OMe—Ph 2-Et—Ph t-Bu RG-101353 2-CH₃, 3-OMe—Ph 3-CH₃, 5-Br—Pht-Bu RG-100767 2-CH₃, 3-OMe—Ph 3-OMe, 5-CH₃—Ph t-Bu RG-100848 2-CH₃,3-OMe—Ph 2-OCH₃-4-Cl—Ph t-Bu RG-101692 2-CH₃, 3-OCF₃—Ph 3-CH₃, 5-CH₃—Pht-Bu RG-100768 2-CH₃, 3-OMe—Ph 2-OCH₃-4-CH₃—Ph t-Bu RG-101585 3-OCH₃,4-CH3—Ph 3-CH₃, 5-CH₃—Ph t-Bu RG-100769 2-CH₃, 3-OMe—Ph 2-OCH₃-4-CH₃—Pht-Bu RG-100394 2-CH₃, 3-OCH₃—Ph 2,6-di-Cl-4-pyridyl t-Bu RG-1005692-CH₃, 3-OMe—Ph 2-NO₂-5-CH₃—Ph t-Bu RG-100929 2-CH₃, 3-OMe—Ph2-F-4-Cl—Ph t-Bu RG-101048 3,4-OCH₂O—Ph 2-Cl-4-F—Ph t-Bu RG-102240 2-Et,3-OMe—Ph 3-CH₃, 5-CH₃—Ph t-Bu RG-101691 2-CH₃, 3-Et—Ph 3-CH₃, 5-CH₃—Pht-Bu RG-101531 3-CH₂CH₂O-4-Ph 3-CH₃, 5-CH₃—Ph t-Bu RG-101382 2-CH₃,3-OMe—Ph 3,5-di-Cl-4-F—Ph t-Bu RG-100448 2-CH₃, 3,4-OCH₂O—Ph 4-F—Ph t-BuRG-100698 2-Et, 3,4-OCH₂O—Ph 2-OCH₃—Ph t-Bu RG-101889 3,4-di-Et—Ph3-CH₃, 5-CH₃—Ph t-Bu RG-100812 2-Et, 3-OMe—Ph 4-F—Ph t-Bu RG-1007252-Et, 3-OMe—Ph 2-OCH₃—Ph t-Bu RG-100524 2-CH₃, 3-OMe—Ph 2-OCH₃-4-F—Pht-Bu RG-100667 2-Et, 3-OCH₃—Ph 2-Cl-6-CH₃-4-pyridyl t-Bu RG-100778 2-Et,3-OMe—Ph 3-OMe, 5-OMe—Ph t-Bu RG-101528 2-I, 3-OMe—Ph 3-CH₃, 5-CH₃—Pht-Bu RG-100492 3,4-ethylenedioxy-Ph 2-OCH₃—Ph t-Bu RG-1018873,4-(CH₂)₄—Ph 3-CH₃, 5-CH₃—Ph t-Bu RG-115496 2-Et, 3-OMe—Ph 2,3-OCH₂O—Pht-Bu RG-100901 2-F, 4-Et—Ph 4-F—Ph t-Bu RG-100699 2-Et, 3-OMe—Ph3,4-methylenedioxy-Ph t-Bu RG-100425 2-CH₃, 4-F—Ph t-Bu3,4-ethylenedioxy-Ph RG-101511 3,4-OCH(CH₃)O—Ph 3-CH₃, 5-CH₃—Ph t-BuRG-101659 2-Et, 3,4-OCH(CH₃)O—Ph 3-CH₃, 5-CH₃—Ph t-Bu RG-100360 2-CH₃,3-OCH₃—Ph t-Bu 3,4-ethylenedioxy-Ph RG-101509 3-OCH(CH₃)CH₂O-4-Ph 3-CH₃,5-CH₃—Ph t-Bu RG-101340 2-Br, 3,4-ethylenedioxy-Ph 3-CH₃, 5-CH₃—Ph t-BuRG-101494 2-Et, 3,4-ethylenedioxy-Ph 3-CH₃, 5-Cl—Ph t-Bu RG-101036 2-Et,3,4-ethylenedioxy-Ph 3-CH₃—Ph t-Bu RG-100690 2-Et, 3,4-ethylenedioxy-Ph2-OCH₃—Ph t-Bu RG-100691 2-Et, 3,4-ethylenedioxy-Ph 3-OCH₃—Ph t-BuRG-101312 3-S—C═N-4-Ph 3-CH₃, 5-CH₃—Ph t-Bu RG-101218 2-Et, 3-OMe—Ph2-OCH₃-4-Cl—Ph t-Bu RG-100779 2-Et, 3-OMe—Ph 2,5-di-OCH₃—Ph t-BuRG-101088 2-CH₃, 3-CH₃, 5-CH₃—Ph t-Bu 4,5-methylenedioxy-Ph RG-1010163-CH₂OCH₂O-4-Ph 3-CH₃, 5-CH₃—Ph t-Bu RG-100216 2-CH₃, 3-OCH₂OCH₂-4-Ph2-OCH₃—Ph t-Bu RG-100574 2-Et, 3-OCH₂OCH₂-4-Ph 4-F—Ph t-Bu RG-1011712-Cl 3-CH₃, 5-CH₃—Ph t-Bu 4,5-methylenedioxy-Ph RG-100620 2,3,6-tri-F—Ph2-Cl-4-F—Ph t-Bu RG-115033 2-Et, 3-OMe—Ph 2,6-F—Ph t-Bu RG-115515 2-Et,3-OMe—Ph 3-F—Ph t-Bu RG-115038 2-Et, 3-OMe—Ph 3-Br—Ph t-Bu RG-1153302-Et, 3-OMe—Ph 2-NO₂—Ph t-Bu RG-115627 2-Et, 3-OMe—Ph 2,3-F—Ph t-BuRG-115329 2-Et, 3-OMe—Ph 3,4,5-tri-OCH₃—Ph t-Bu RG-115088 2-Et, 3-OMe—Ph3-CF₃, 4-F—Ph t-Bu RG-115327 2-Et, 3-OMe—Ph 3-CN—Ph t-Bu RG-1155342-Vinyl, 3-OMe—Ph 2,4-di-Cl-5-F—Ph t-Bu RG-115046 2-Et, 3-OCH₂OCH₂-4-PhPh t-Bu RG-115025 2-Et, 3-OMe—Ph 3-CH₃, 5-CH₃—Ph —C(CH₃)₂C(O)OEtRG-115143 2-Et, 3-OMe—Ph 3-CH₃, 5-CH₃—Ph —C(CH₃)₂CH₂OH RG-115407 2-Et,3-OMe—Ph 3-CH₃, 5-CH₃—Ph —C(CH₃)₂CHO RG-115006 2-Et, 3-OMe—Ph 3-CH₃,5-CH₃—Ph —C(CH₃)₂CH₂OCH₃ RG-115258 2-Et, 3-OMe—Ph 3-CH₃, 5-CH₃—Ph—C(CH₃)₂CH═NOH RG-115378 2-NH₂, 3-OMe—Ph 3-CH₃, 5-CH₃—Ph t-Bu RG-1152232-Et, 3-OMe—Ph 3-CH₂OAc, 5-CH₃—Ph t-Bu RG-115310 2-Et, 3-OMe—Ph 3-CH₃,5-CH₃—Ph —C(CH₃)₂CH₂OC(O)CH₃ RG-115567 2-CH₃, 3-OH—Ph 2,3,4-F—Ph t-BuRG-115443 2-CH₃, 3-OH—Ph 3-Cl-5-OCH₃-4-pyridyl t-Bu RG-115261 2-CH₃,3-OH—Ph 2,6-di-Cl-4-pyridyl t-Bu RG-115595 2-CH₃, 3-OH—Ph3-OCH₃-4-pyridyl t-Bu RG-115220 2-CH₃, 3-OH—Ph 3,5-di-OCH₃-4-CH₃—Ph t-BuRG-115102 2-CH₃, 2-OCH₃—Ph t-Bu 3-CH₂CH₂CH₂O-4-Ph RG-115302 2-Et,3-OMe—Ph 2,4-di-Cl-5-F—Ph t-Bu RG-115539 2-CH₃, 2,4-di-Cl-5-F—Ph t-Bu3-CH₂CH₂CH₂O-4-Ph RG-115499 2-CH₃, 2-F, 5-CH₃—Ph t-Bu 3-CH₂CH₂CH₂O-4-PhRG-115055 2-CH₃, 3,5-di-OCH₃-4-CH₃—Ph t-Bu 3-CH₂CH₂CH₂O-4-Ph RG-1155082-Et, 3,4-ethylenedioxy-Ph 2,5-F—Ph t-Bu RG-115580 2-Et,3,4-ethylenedioxy-Ph 2,3,4-F—Ph t-Bu RG-115337 2-Et,3,4-ethylenedioxy-Ph 2,3,4,5--Ph t-Bu RG-115280 2-Et,3,4-ethylenedioxy-Ph 3-CF₃-4-F—Ph t-Bu RG-115297 2-Et,3,4-ethylenedioxy-Ph 2,6-di-Cl-4-pyridyl t-Bu RG-115244 2-Et,3,4-ethylenedioxy-Ph 2-OCH₃—Ph t-Bu RG-115684 2-Et, 3,4-ethylenedioxy-Ph2,4-di-Cl-5-F—Ph t-Bu RG-115514 2-Et, 3,4-ethylenedioxy-Ph 2-F, 4-Cl—Pht-Bu RG-115557 2-CH₃, 3-OAc—Ph 3,5-di-OCH₃-4-CH₃—Ph t-Bu RG-115253 2-Et,3-OMe—Ph 2-OCH₃-5-Cl—Ph t-Bu RG-115085 2-Et, 3,4-OCH₂O—Ph 2-OCH₃-4-Cl—Pht-Bu RG-115551 2-CH₃, 2-OCH₃-5-Cl—Ph t-Bu 3-CH₂CH₂CH₂O-4-Ph RG-1151622-Et, 3-OMe—Ph 2-NO₂-5-CH₃—Ph t-Bu RG-115647 2-Et, 3-OMe—Ph2-NO₂-4-Cl—Ph t-Bu RG-115257 2-Et, 3-OMe—Ph 2-NO₂-5-Cl—Ph t-Bu RG-1156642-CH₃, 2-NO₂-5-CH₃—Ph t-Bu 3-CH₂CH₂CH₂O-4-Ph RG-115171Benzo[1,2,5]oxadiazole- 2-OCH₃-4-Cl—Ph t-Bu 5-yl RG-115480 2-Vinyl,3-OMe—Ph 2-Cl, 5-NO₂—Ph t-Bu RG-115095 2-Vinyl, 3-OMe—Ph 2-OCH₃-4-Cl—Pht-Bu RG-115106 2-Et, 3-OCH₃—Ph 1-methyl-1H-indole-2-yl t-Bu RG-1151302-Et, 3,4-ethylenedioxy-Ph 3,5-di-OCH₃-4-CH₃—Ph t-Bu RG-115532 2-Cl,3-CH₂CH₂CH₂O-4-Ph 3-CH₃, 5-CH₃—Ph t-Bu RG-115167 2-F, 4-Et—Ph 3-NO₂—Pht-Bu RG-115269 2-F, 4-Et—Ph 3-OCH₃—Ph t-Bu RG-115441 2-Cl,3-CH₂CH₂CH₂O-4-Ph 3,5-di-OCH₃-4-CH₃—Ph t-Bu RG-115128 2-F, 4-Et—Ph2,6-di-Cl-4-pyridyl t-Bu RG-115077 2-F, 4-Et—Ph 3,5-di-OCH₃-4-CH₃—Pht-Bu RG-115259 2-F, 4-Et—Ph 3,4,5-F—Ph t-Bu RG-115674 2-F, 4-Et—Ph3-CH₃—Ph t-Bu RG-115422 2-F, 4-Et—Ph 2-OCH₃—Ph t-Bu RG-115086 2-F,4-Et—Ph 2-NO₂-5-F—Ph t-Bu RG-115592 2-F, 4-Et—Ph 2-OCH₂CF₃, 5-OCH₃—Pht-Bu RG-115112 2-F, 4-Et—Ph 2-Cl-6-CH₃-4-pyridyl t-Bu RG-115050 2-F,4-Et—Ph 2,6-di-OCH₃-3-pyridyl t-Bu RG-115689 3-NH—C═C-4-Ph 3-CH₃,5-CH₃—Ph t-Bu RG-115199 2-Et, 3-OMe—Ph 2-S(O)CH₃—Ph t-Bu RG-1153523,4-OCF₂O—Ph 2-NO₂—Ph t-Bu RG-115256 3,4-OCF₂O—Ph 3-CH₃, 5-CH₃—Ph t-BuRG-115683 3,4-OCF₂O—Ph 5-OCH₃—Ph t-Bu RG-115648 2-Et, 3-OMe—Ph 3-Br—Ph—C(CH₃)₂CN RG-115306 2-CH₂OMe, 3-OMe—Ph 3,5-di-Cl—Ph t-Bu RG-1156252-Et, 3-OMe—Ph 3-CH═NOH, 5-CH₃—Ph t-Bu RG-115429 2-Et, 3-OMe—Ph3-CH═NNHCONH₂, 5-CH₃—Ph t-Bu RG-115613 2-Et, 3-OMe—Ph 3-CH═NNHCOCONH₂,t-Bu 5-CH₃—Ph RG-115043 2-Et, 3-OMe—Ph 3-CH₃, 5-CH₃—Ph —C(CH₃)₂CNRG-115690 2-Et, 3-OMe—Ph 3,5-di-OCH₃-4-CH₃—Ph —C(CH₃)₂CN RG-115065 2-Et,3-OCH₂OCH₂-4—Ph 2-OCH₃—Ph t-Bu RG-115229 2-CH₃, 2,4,5-F—Ph t-Bu3-CH₂CH₂CH₂O-4-Ph RG-115575 2-CH₃, 3,4,5-F—Ph t-Bu 3-CH₂CH₂CH₂O-4-PhRG-115278 2-CH₃, 3-F—Ph t-Bu 3-CH₂CH₂CH₂O-4-Ph RG-115260 2-Et,3,4-OCH₂O—Ph 3-CF₃—Ph t-Bu RG-115118 2-CH₃, 4-F—Ph t-Bu3-CH₂CH₂CH₂O-4-Ph RG-115416 2-CH₃, 3,4-F—Ph t-Bu 3-CH₂CH₂CH₂O-4-PhRG-115207 2-CH₃, 3,5-di-F—Ph t-Bu 3-CH₂CH₂CH₂O-4-Ph RG-115518 2-CH₃,2,3,4,5-tetra-F—Ph t-Bu 3-CH₂CH₂CH₂O-4-Ph RG-115611 2-Et,3-OCH₂OCH₂-4—Ph 4-CH₃—Ph t-Bu RG-115191 2-Et, 3-OMe—Ph3,5-di-OCH₃-4-OAc—Ph t-Bu RG-115116 2-Et, 3-OMe—Ph 3,5-di-OCH₃—OH—Pht-Bu RG-115637 2-CH₃, 3,5-di-OCH₃-4-CH₃—Ph t-Bu 3,4-ethylenedioxy-PhRG-115517 2-CH₃, 2,6-di-OCH₃-3-pyridyl t-Bu 3,4-ethylenedioxy-PhRG-115536 2-CH₃, 2,6-di-Cl-4-pyridyl t-Bu 3,4-ethylenedioxy-Ph RG-1153502-CH₃, 3-F—Ph t-Bu 3,4-ethylenedioxy-Ph RG-115169 2-CH₃, 3-CF₃, 5-F—Pht-Bu 3,4-ethylenedioxy-Ph RG-115384 2-CH₃, 2-NO₂-5-CH₃—Ph t-Bu3,4-ethylenedioxy-Ph RG-115783 2-ethyl, 3-methoxy 4,6-dimethyl-pyridylt-Bu RG-115856 2-CH₃, 3,5-di-CH₃—Ph —CH(Et)C(CH₃)₃ 3,4-ethylenedioxy-PhRG-115857 2-CH₃, 3,5-di-OCH₃-4-CH₃—Ph —CH(Et)C(CH₃)₃3,4-ethylenedioxy-Ph RG-115864 2-CH₃, 3,5-di-CH₃—Ph —CH(n-Pr)C(CH₃)₃3,4-ethylenedioxy-Ph RG-115865 2-CH₃, 3,5-di-OCH₃-4-CH₃—Ph—CH(n-Pr)C(CH₃)₃ 3,4-ethylenedioxy-Ph RG-115858 2-CH₂CH₃, 3,5-di-CH₃—Ph—CH(Et)C(CH₃)₃ 3,4-ethylenedioxy-Ph RG-115859 2-CH₂CH₃,3,5-di-OCH3-4-CH3—Ph —CH(Et)C(CH₃)₃ 3,4-ethylenedioxy-Ph RG-1158662-CH₂CH₃, 3,5-di-CH₃—Ph —CH(n-Pr)C(CH₃)₃ 3,4-ethylenedioxy-Ph RG-1158672-CH₂CH₃, 3,5-di-OCH₃-4-CH₃—Ph —CH(n-Pr)C(CH₃)₃ 3,4-ethylenedioxy-PhRG-115834 2-CH₃, 3-OCH₃—Ph 2-methoxy-6-trifluoromethyl-3- —C(CH₃)₃pyridyl RG-115835 2-CH₃, 3-OCH₃—Ph 1-methyl-2-oxo-6-trifluoromethyl-—C(CH₃)₃ 3-pyridyl RG-115849 2-CH₃, 3-OCH₃—Ph2,6-dimethoxy-4-pyrimidinyl —C(CH₃)₃ RG-115850 2-CH₃, 3-OCH₃—Ph3,6-dimethoxy-4-pyridazinyl —C(CH₃)₃ RG-115861 2-CH₃, 3-OCH₃—Ph3,6-dichloro-4-pyridazinyl —C(CH₃)₃ RG-115862 2-CH₃, 3-OCH₃—Ph4-pyridazinyl —C(CH₃)₃ RG-115863 2-CH₃, 3-OCH₃—Ph3-oxo-6-methoxy-4-pyridazinyl —C(CH₃)₃ (or regioisomer) RG-115819 2-CH₃,3-OCH₃—Ph 3,5-di-CH₃—Ph —CH(Et)C(CH₃)₃ RG-115820 2-CH₃, 3-OCH₃—Ph3,5-di-OCH₃-4-CH₃—Ph —CH(Et)C(CH₃)₃ RG-115823 2-CH₃, 3-OCH₃—Ph3,5-di-CH₃—Ph —CH(n-Pr)C(CH₃)₃ RG-115824 2-CH₃, 3-OCH₃—Ph3,5-di-OCH₃-4-CH₃—Ph —CH(n-Pr)C(CH₃)₃ RG-115832 2-CH₂CH₃, 3-OCH₃—Ph3,5-di-CH₃—Ph —CH(Et)C(CH₃)₃ RG-115831 2-CH₂CH₃, 3-OCH₃—Ph3,5-di-OCH₃-4-CH₃—Ph —CH(Et)C(CH₃)₃ RG-115830 2-CH₂CH₃, 3-OCH₃—Ph3,5-di-CH₃—Ph —CH(n-Pr)C(CH₃)₃ RG-115829 2-CH₂CH₃, 3-OCH₃—Ph3,5-di-OCH₃-4-CH₃—Ph —CH(n-Pr)C(CH₃)₃ RG-103309 2-CH₃, 3-OCH₃—Ph3,5-di-CH₃—Ph —CH(Et)C(CH₃)₃ RG-115595 2-CH₃, 3-OH—Ph 3-OCH₃-4-pyridyl—C(CH₃)₃ RG-100021 4-CH(OH)CH₃—Ph 3,5-di-(CH₂OH)—Ph —C(CH₃)₃ RG-1151992-CH₂CH₃, 3-OCH₃—Ph 2-S(O)CH₃—Ph —C(CH₃)₃ RG-100150 4-C(O)CH₃—Ph3,5-di-CO₂H—Ph —C(CH₃)₃ RG-115517 2-CH₃, 2,6-di-OCH₃-3-pyridyl —C(CH₃)₃3,4-ethylenedioxy-Ph RG-115280 2-CH₂CH₃, 3-CF₃-4-F-pyridyl —C(CH₃)₃3,4-ethylenedioxy-Ph RG-101523 2-F, 4-CH₂CH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₃RG-115555 2-CH₂CH₃, 3-OCH₃—Ph 2-SO₃H—Ph —C(CH₃)₃ RG-102408 2-CH₃,3,5-di-CH₃—Ph —C(CH₃)₃ 3-CH₂CH₂CH₂O-4-Ph RG-103451 4-CH₂CH₃—Ph3,5-di-CH₃—Ph —CH(CH₃)C(CH₃)₃ RG-101036 2-CH₂CH₃, 3-CH₃—Ph —C(CH₃)₃3,4-ethylenedioxy-Ph RG-103361 2,3-di-CH₃—Ph Ph —CH(Et)(n-Bu) RG-1040742,3-di-CH₃—Ph 3-CH₃—Ph —CH(Et)(t-Bu) RG-115009 2-CH₃, 3,5-di-OCH₃,4-OH—Ph —C(CH₃)₃ 3,4-ethylenedioxy-Ph RG-115068 2-F, 3-CH₂OCH₂O-4-Ph3,5-di-CH₃—Ph —C(CH₃)₃ RG-115064 2-CH₃, 2-S(O)CH₃—Ph —C(CH₃)₃3,4-ethylenedioxy-Ph RG-115092 2-CH₃, 3,5-di-OCH₃, 4-CH₃—Ph —C(CH₃)₂CN3,4-ethylenedioxy-Ph RG-115311 2-CH₂CH₃-3-OCH₃—Ph 6-CH₃-2-pyridyl-—C(CH₃)₃ RG-115609 2-CH₃, 2-NO₂-3,5-di-OCH₃, 4-CH₃—Ph —C(CH₃)₃3,4-ethylenedioxy-Ph RG-102317 2-CH₃, 3,5-di-CH₃—Ph —C(CH₃)₃3,4-ethylenedioxy-Ph RG-102125 4-CH₂CH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₃RG-102398 2-CH₃-3-OCH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₃ RG-115836 4-CH₂CH₃—Ph3,5-di-CH₃—Ph —CH(Et)(t-Bu) RG-115837 4-CH₂CH₃—Ph 2-OCH₃-3-pyridyl—CH(Et)(t-Bu) RG-115840 4-CH₂CH₃—Ph 3,5-di-CH₃—Ph —CH(n-Bu)(t-Bu)RG-115841 4-CH₂CH₃—Ph 3,5-di-OCH₃, 4-CH₃—Ph —CH(n-Bu)(t-Bu) RG-1158424-CH₂CH₃—Ph 2-OCH₃-3-pyridyl —CH(n-Bu)(t-Bu) RG-115846 4-CH₂CH₃—Ph3,5-di-CH₃—Ph —CH(Ph)(t-Bu) RG-115847 4-CH₂CH₃—Ph 3,5-di-OCH₃, 4-CH₃—Ph—CH(Ph)(t-Bu) RG-115848 4-CH₂CH₃—Ph 2-OCH₃-3-pyridyl —CH(Ph)(t-Bu)RG-115719 2-CH₂CH₃, 3-OCH₃—Ph 5-benzimidazolyl —C(CH₃)₃ RG-1157182-CH₂CH₃, 3-OCH₃—Ph 1-(or 3-)trityl-5-benzimidazolyl —C(CH₃)₃ RG-1157212-CH₂CH₃, 3-OCH₃—Ph 5-methyl-1-phenyl-1H-pyrazole-3- —C(CH₃)₃ ylRG-115716 2-CH₂CH₃, 3-OCH₃—Ph 3-chloro-6-methylsulfanyl-pyrazine-—C(CH₃)₃ 2-yl RG-115723 2-CH₂CH₃, 3-OCH₃—Ph 1H-indazole-3-yl —C(CH₃)₃RG-115722 2-CH₂CH₃, 3-OCH₃—Ph 1-trityl-1H-indazole-3-yl —C(CH₃)₃RG-115717 2-CH₂CH₃, 3-OCH₃—Ph 5-methoxycarbonyl-2-pyridyl —C(CH₃)₃RG-115550 2-CH₂CH₃, 3-OCH₃—Ph pyrazine-2-yl —C(CH₃)₃ RG-115665 2-CH₂CH₃,3-OCH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₂CH₂OSi(CH₃)2tBu RG-115511 2-CH₂CH₃,3-OCH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₂CH═NCH₂CH₂OH RG-115653 2-CH₂CH₃,3-OCH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₂CH═NNHC(O)NH₂ RG-115597 2-CH₂CH₃,3-OCH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₂CH═NNHC(O)C(O)NH₂ RG-115044 2-CH₂CH₃,3-OCH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₂COOH RG-115172 2-CH₂(O)CH₃,3,5-di-CH₃—Ph —C(CH₃)₃ 3-OCH₃—Ph RG-115408 2-CH₂(O)CH₃, 3,5-di-CH₃—Ph—C(CH₃)₃ 3-OCH3—Ph RG-115497 2-CH₂NMe₂, 3-OCH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₃RG-115079 2-CH₂NHCH₃, 3-OCH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₃ RG-1020212-CH═CH₂, 3-OCH₃—Ph— 3,5-di-CH₃—Ph —C(CH₃)₃ RG-115117 2-CH₂OMe,3-OCH₃—Ph— 3,5-di-CH₃—Ph —C(CH₃)₃ RG-115358 2-CH₂SCH₃, 3-OCH₃—Ph3,5-di-CH₃—Ph —C(CH₃)₃ RG-115003 2-CH₂OCH₂CH═CH₂, 3,5-di-CH₃—Ph —C(CH₃)₃3-OCH₃—Ph RG-115490 2-CH₂Cl, 3-OCH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₃ RG-1153712-CH₂OH, 3-OCH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₃ RG-115225 2-CH₂OAc, 3-OCH₃—Ph3,5-di-CH₃—Ph —C(CH₃)₃ RG-115160 2-CH₂F, 3-OCH₃—Ph 3,5-di-CH₃—Ph—C(CH₃)₃ RG-115851 2-CH₃, 3-OCH₃ 3,5-di-CH₃ —CH(n-Bu)(t-Bu) RG-1158522-CH₃, 3-OCH₃ 3,5-di-OCH₃, 4-CH₃ —CH(n-Bu)(t-Bu) RG-115091 2-CH₂CH₃,3-OCH₃—Ph 5-Methyl-pyrazine-2-yl- —C(CH₃)₃

Because the compounds of the general formula of the present inventionmay contain a number of stereogenic carbon atoms, the compounds mayexist as enantiomers, diastereomers, stereoisomers, or their mixtures,even if a stereogenic center is explicitly specified.

The present invention also pertains to a process for the preparation ofa compound of formula (IV) comprising the steps of:

-   -   i reacting a compound of formula (I) with a base selected from        NaH, KH, or an amide MNR^(a)R^(b) to produce a product II,        wherein M is Li, Na, or K, and R^(a) and R^(b) are independently        (C₁-C₆)alkyl or phenyl; and

-   -   ii reacting the product (II) of step (i) with a compound of        formula (III) wherein R is phenyl substituted with three to five        of the same or different chloro, fluoro, or trifluoromethyl;

wherein

A and B are independently

-   -   (a) unsubstituted or substituted phenyl wherein the substituents        are independently 1 to 5H; halo; nitro; cyano; amino        (—NR^(a)R^(b)); alkylaminoalkyl (—(CH₂)_(n)NR^(a)R^(b));        (C₁-C₆)alkyl; (C₁-C₆)haloalkyl; (C₁-C₆)cyanoalkyl;        (C₁-C₆)alkoxy; phenoxy; (C₁-C₆)haloalkoxy;        (C₁-C₆)alkoxy(C₁-C₆)alkyl; (C₁-C₆)alkenyloxy(C₁-C₆)alkyl;        (C₁-C₆)alkoxy(C₁-C₆)alkoxy; (C₂-C₆)alkenyl optionally        substituted with halo, cyano, (C₁-C₄)alkyl, or (C₁-C₄)alkoxy;        (C₂-C₆)alkynyl optionally substituted with halo or (C₁-C₄)alkyl;        formyl; (C₁-C₆)haloalkylcarbonyl; benzoyl;        (C₁-C₆)alkoxycarbonyl; (C₁-C₆)haloalkoxycarbonyl;        (C₁-C₆)alkanoyloxy (—OCOR^(a)); carboxamido (—CONR^(a)R^(b));        amido (—NR^(a)COR^(b)); alkoxycarbonylamino        (—N(CH₂)_(n)CO₂R^(b)); alkylaminocarbonylamino        (—N(CH₂)_(n)CONR^(b)R^(c)); (C₁-C₆)alkylthio; sulfamido        (—SO₂NR^(a)R^(b)); or unsubstituted or substituted phenyl        wherein the substituents are independently 1 to 3 halo, nitro,        (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or (—NR^(a)R^(b)); or when one or        both of two adjacent positions on the phenyl ring are        substituted, the attached atoms may form the phenyl-connecting        termini of a linkage selected from the group consisting of        (—OCH₂O—), (—OCH(CH₃)O—), (—OCH₂CH₂O—), (—OCH(CH₃)CH₂O—),        (—S—CH═N—), (—CH₂OCH₂O—), (—O(CH₂)₃—), (═N—O—N═), (—C═CH—NH—),        (—OCF₂O—), (—NH—CH═N—), (—CH₂CH₂O—), and (—(CH₂)₄—); or    -   (b) unsubstituted 5- or 6-membered heterocycle or substituted 5        or 6-membered heterocycle having 1-3 nitrogen atoms where the        substituents are from one to four of the same or different halo;        nitro; (C₁-C₆)alkyl; (C₁-C₆)alkeyl; (C₁-C₆)alkoxy;        (C₁-C₆)thioalkoxy; (C₁-C₆)alkoxycarbonyl; (C₁-C₆)carbocyalkyl;        —CONR^(a)R^(b); amino (—NR^(a)R^(b)); haloalkyl including —CF₃;        -trialkylsilyl (—SiR^(a)R^(b)R^(c)); trityl (C(Ph)₃); or        unsubstituted or substituted phenyl wherein the substituents are        independently 1 to 3 halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl,        or (—NR^(a)R^(b)); or when two adjacent positions are        substituted, these positions may form a benzo ring fusion; and

E is phenyl, or unsubstituted or substituted (C₁-C₁₀) straight orbranched alkyl wherein the substituents are independently 1-4 cyano;halo; (C₅-C₆)cycloalkyl; phenyl; (C₂-C₃)alkenyl; (C₁-C₆)alkoxy;(C₁-C₆)alkoxycarbonyl; (C₁-C₆)alkanoyloxy (—OCOR^(a)); formyl;(C₁-C₆)trialkylsilyloxy having independently the stated number of carbonatoms in each alkyl group; or —C═N—OR^(a);

wherein R^(a), R^(b), and R^(c) are independently (C₁-C₆)alkyl orphenyl, and n=1-4.

DEFINITIONS

When an R^(x) group is specified, wherein x represents a letter a-g, andthe same R^(x) group is also specified with an alkyl group chain lengthsuch as “(C₁-C₃)”, it is understood that the specified chain lengthrefers only to the cases where R^(x) may be alkyl, and does not pertainto cases where R^(x) may be a non-alkyl group, such as H or aryl.

The term “alkyl” includes both branched and straight chain alkyl groups.Typical alkyl groups include, for example, methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl,isopentyl, n-hexyl, n-heptyl, isooctyl, nonyl, and decyl.

The term “halo” refers to fluoro, chloro, bromo or iodo.

The term “haloalkyl” refers to an alkyl group substituted with one ormore halo groups such as, for example, chloromethyl, 2-bromoethyl,3-iodopropyl, trifluoromethyl, and perfluoropropyl.

The term “cycloalkyl” refers to a cyclic aliphatic ring structure,optionally substituted with alkyl, hydroxy, or halo, such ascyclopropyl, methylcyclopropyl, cyclobutyl, 2-hydroxycyclopentyl,cyclohexyl, and 4-chlorocyclohexyl.

The term “hydroxyalkyl” refers to an alkyl group substituted with one ormore hydroxy groups such as, for example, hydroxymethyl and2,3-dihydroxybutyl.

The term “alkylsulfonyl” refers to a sulfonyl moiety substituted with analkyl group such as, for example, mesyl, and n-propylsulfonyl.

The term “alkenyl” refers to an ethylenically unsaturated hydrocarbongroup, straight or branched chain, having 1 or 2 ethylenic bonds suchas, for example, vinyl, allyl, 1-butenyl, 2-butenyl, isopropenyl, and2-pentenyl.

The term “haloalkenyl” refers to an alkenyl group substituted with oneor more halo groups. The term “alkynyl” refers to an unsaturatedhydrocarbon group, straight or branched, having 1 or 2 acetylenic bondssuch as, for example, ethynyl and propargyl.

The term “alkylcarbonyl” refers to an alkylketo functionality, forexample acetyl, n-butyryl and the like.

The term “heterocyclyl” or “heterocycle” refers to an unsubstituted orsubstituted; saturated, partially unsaturated, or unsaturated 5 or6-membered ring containing one, two or three heteroatoms, preferably oneor two heteroatoms independently selected from the group consisting ofoxygen, nitrogen and sulfur. Examples of heterocyclyls include, forexample, pyridyl, thienyl, furyl, pyrimidinyl, pyrazinyl, quinolinyl,isoquinolinyl, pyrrolyl, indolyl, tetrahydrofuryl, pyrrolidinyl,piperidinyl, tetrahydropyranyl, morpholinyl, piperazinyl, dioxolanyl,and dioxanyl.

The term “alkoxy” includes both branched and straight chain alkyl groupsattached to a terminal oxygen atom. Typical alkoxy groups include, forexample, methoxy, ethoxy, n-propoxy, isopropoxy, and tert-butoxy.

The term “haloalkoxy” refers to an alkoxy group substituted with one ormore halo groups such as, for example chloromethoxy, trifluoromethoxy,difluoromethoxy, and perfluoroisobutoxy.

The term “alkylthio” includes both branched and straight chain alkylgroups attached to a terminal sulfur atom such as, for examplemethylthio.

The term “haloalkylthio” refers to an alkylthio group substituted withone or more halo groups such as, for example trifluoromethylthio.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group such as, for example, isopropoxymethyl.

“Silica gel chromatography” refers to a purification method wherein achemical substance of interest is applied as a concentrated sample tothe top of a vertical column of silica gel or chemically-modified silicagel contained in a glass, plastic, or metal cylinder, and elution fromsuch column with a solvent or mixture of solvents.

“Flash chromatography” refers to silica gel chromatography performedunder air, argon, or nitrogen pressure typically in the range of 10 to50 psi.

“Gradient chromatography” refers to silica gel chromatography in whichthe chemical substance is eluted from a column with a progressivelychanging composition of a solvent mixture.

“Rf” is a thin layer chromatography term which refers to the fractionaldistance of movement of a chemical substance of interest on a thin layerchromatography plate, relative to the distance of movement of theeluting solvent system.

“Parr hydrogenator” and “Parr shaker” refer to apparatus available fromParr Instrument Company, Moline Ill., which are designed to facilitatevigorous mixing of a solution containing a chemical substance ofinterest with an optional solid suspended catalyst and a pressurized,contained atmosphere of a reactant gas. Typically, the gas is hydrogenand the catalyst is palladium, platinum, or oxides thereof deposited onsmall charcoal particles. The hydrogen pressure is typically in therange of 30 to 70 psi.

“Dess-Martin reagent” refers to(1,1,1-triacetoxy)-1,1-dihydro-1,2-benziodoxol-3(1H)-one as a solutionin dichloromethane available from Acros Organics/Fisher ScientificCompany, L.L.C.

“PS-NMM” refers to a —SO₂NH(CH₂)₃-morpholine functionalized polystyreneresin available from Argonaut Technologies, San Carlos, Calif.

“AP-NCO” refers to an isocyante-functionalized resin available fromArgonautTechnologies, San Carlos, Calif.

“AP-trisamine” refers to a polystyrene-CH₂NHCH₂CH₂NH(CH₂CH₂NH₂)₂ resinavailable from Argonaut Technologies, San Carlos, Calif.

The term “isolated” for the purposes of the present invention designatesa biological material (nucleic acid or protein) that has been removedfrom its original environment (the environment in which it is naturallypresent). For example, a polynucleotide present in the natural state ina plant or an animal is not isolated, however the same polynucleotideseparated from the adjacent nucleic acids in which it is naturallypresent, is considered “isolated”. The term “purified” does not requirethe material to be present in a form exhibiting absolute purity,exclusive of the presence of other compounds. It is rather a relativedefinition.

A polynucleotide is in the “purified” state after purification of thestarting material or of the natural material by at least one order ofmagnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.

A “nucleic acid” is a polymeric compound comprised of covalently linkedsubunits called nucleotides. Nucleic acid includes polyribonucleic acid(RNA) and polydeoxyribonucleic acid (DNA), both of which may besingle-stranded or double-stranded. DNA includes but is not limited tocDNA, genomic DNA, plasmids DNA, synthetic DNA, and semi-synthetic DNA.DNA may be linear, circular, or supercoiled.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

The term “fragment” will be understood to mean a nucleotide sequence ofreduced length relative to the reference nucleic acid and comprising,over the common portion, a nucleotide sequence identical to thereference nucleic acid. Such a nucleic acid fragment according to theinvention may be, where appropriate, included in a larger polynucleotideof which it is a constituent. Such fragments comprise, or alternativelyconsist of, oligonucleotides ranging in length from at least 6, 8, 9,10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51,54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200,300, 500, 720, 900, 1000 or 1500 consecutive nucleotides of a nucleicacid according to the invention.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

A “gene” refers to an assembly of nucleotides that encode a polypeptide,and includes cDNA and genomic DNA nucleic acids. “Gene” also refers to anucleic acid fragment that expresses a specific protein or polypeptide,including regulatory sequences preceding (5′ non-coding sequences) andfollowing (3′ non-coding sequences) the coding sequence. “Native gene”refers to a gene as found in nature with its own regulatory sequences.“Chimeric gene” refers to any gene that is not a native gene, comprisingregulatory and/or coding sequences that are not found together innature. Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature. A chimericgene may comprise coding sequences derived from different sources and/orregulatory sequences derived from different sources. “Endogenous gene”refers to a native gene in its natural location in the genome of anorganism. A “foreign” gene or “heterologous” gene refers to a gene notnormally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Heterologous” DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

The term “genome” includes chromosomal as well as mitochondrial,chloroplast and viral DNA or RNA.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., 1989 infra). Hybridization andwashing conditions are well known and exemplified in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor(1989), particularly Chapter 11 and Table 11.1 therein (entirelyincorporated herein by reference). The conditions of temperature andionic strength determine the “stringency” of the hybridization.

Stringency conditions can be adjusted to screen for moderately similarfragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. For preliminaryscreening for homologous nucleic acids, low stringency hybridizationconditions, corresponding to a T_(m) of 55°, can be used, e.g., 5×SSC,0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5%SDS). Moderate stringency hybridization conditions correspond to ahigher T_(m), e.g., 40% formamide, with 5× or 6×SCC. High stringencyhybridization conditions correspond to the highest T_(m), e.g., 50%formamide, 5× or 6×SCC.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The term “complementary” is usedto describe the relationship between nucleotide bases that are capableof hybridizing to one another. For example, with respect to DNA,adenosine is complementary to thymine and cytosine is complementary toguanine. Accordingly, the instant invention also includes isolatednucleic acid fragments that are complementary to the complete sequencesas disclosed or used herein as well as those substantially similarnucleic acid sequences.

In a specific embodiment of the invention, polynucleotides are detectedby employing hybridization conditions comprising a hybridization step atT_(m) of 55° C., and utilizing conditions as set forth above. In apreferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) is 63° C.; in an even more preferred embodiment,the T_(m) is 65° C.

Post-hybridization washes also determine stringency conditions. One setof preferred conditions uses a series of washes starting with 6×SSC,0.5% SDS at room temperature for 15 minutes (min), then repeated with2×SSC, 0.5% SDS at 45° C. for 30 minutes, and then repeated twice with0.2×SSC, 0.5% SDS at 50° C. for 30 minutes. A more preferred set ofstringent conditions uses higher temperatures in which the washes areidentical to those above except for the temperature of the final two 30min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Anotherpreferred set of highly stringent conditions uses two final washes in0.1×SSC, 0.1% SDS at 65° C. Hybridization requires that the two nucleicacids comprise complementary sequences, although depending on thestringency of the hybridization, mismatches between bases are possible.

The appropriate stringency for hybridizing nucleic acids depends on thelength of the nucleic acids and the degree of complementation, variableswell known in the art. The greater the degree of similarity or homologybetween two nucleotide sequences, the greater the value of T_(m) forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA: RNA, DNA: RNA, DNA: DNA. For hybrids ofgreater than 100 nucleotides in length, equations for calculating T_(m)have been derived (see Sambrook et al., supra, 9.50-0.51). Forhybridization with shorter nucleic acids, i.e., oligonucleotides, theposition of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (see Sambrook et al., supra,11.7-11.8).

In a specific embodiment of the invention, polynucleotides are detectedby employing hybridization conditions comprising a hybridization step inless than 500 mM salt and at least 37 degrees Celsius, and a washingstep in 2XSSPE at least 63 degrees Celsius. In a preferred embodiment,the hybridization conditions comprise less than 200 mM salt and at least37 degrees Celsius for the hybridization step. In a more preferredembodiment, the hybridization conditions comprise 2XSSPE and 63 degreesCelsius for both the hybridization and washing steps.

In one embodiment, the length for a hybridizable nucleic acid is atleast about 10 nucleotides. Preferable a minimum length for ahybridizable nucleic acid is at least about 15 nucleotides; morepreferably at least about 20 nucleotides; and most preferably the lengthis at least 30 nucleotides. Furthermore, the skilled artisan willrecognize that the temperature and wash solution salt concentration maybe adjusted as necessary according to factors such as length of theprobe.

The term “probe” refers to a single-stranded nucleic acid molecule thatcan base pair with a complementary single stranded target nucleic acidto form a double-stranded molecule.

As used herein, the term “oligonucleotide” refers to a nucleic acid,generally of at least 18 nucleotides, that is hybridizable to a genomicDNA molecule, a cDNA molecule, a plasmid DNA or an mRNA molecule.Oligonucleotides can be labeled, e.g., with ³²P-nucleotides ornucleotides to which a label, such as biotin, has been covalentlyconjugated. A labeled oligonucleotide can be used as a probe to detectthe presence of a nucleic acid. Oligonucleotides (one or both of whichmay be labeled) can be used as PCR primers, either for cloning fulllength or a fragment of a nucleic acid, or to detect the presence of anucleic acid. An oligonucleotide can also be used to form a triple helixwith a DNA molecule. Generally, oligonucleotides are preparedsynthetically, preferably on a nucleic acid synthesizer. Accordingly,oligonucleotides can be prepared with non-naturally occurringphosphoester analog bonds, such as thioester bonds, etc.

A “primer” is an oligonucleotide that hybridizes to a target nucleicacid sequence to create a double stranded nucleic acid region that canserve as an initiation point for DNA synthesis under suitableconditions. Such primers may be used in a polymerase chain reaction.

“Polymerase chain reaction” is abbreviated PCR and means an in vitromethod for enzymatically amplifying specific nucleic acid sequences. PCRinvolves a repetitive series of temperature cycles with each cyclecomprising three stages: denaturation of the template nucleic acid toseparate the strands of the target molecule, annealing a single strandedPCR oligonucleotide primer to the template nucleic acid, and extensionof the annealed primer(s) by DNA polymerase. PCR provides a means todetect the presence of the target molecule and, under quantitative orsemi-quantitative conditions, to determine the relative amount of thattarget molecule within the starting pool of nucleic acids.

“Reverse transcription-polymerase chain reaction” is abbreviated RT-PCRand means an in vitro method for enzymatically producing a target cDNAmolecule or molecules from an RNA molecule or molecules, followed byenzymatic amplification of a specific nucleic acid sequence or sequenceswithin the target cDNA molecule or molecules as described above. RT-PCRalso provides a means to detect the presence of the target molecule and,under quantitative or semi-quantitative conditions, to determine therelative amount of that target molecule within the starting pool ofnucleic acids.

A DNA “coding sequence” is a double-stranded DNA sequence that istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.“Suitable regulatory sequences” refer to nucleotide sequences locatedupstream (5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, polyadenylation recognition sequences, RNAprocessing site, effector binding site and stem-loop structure. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl)terminus. A coding sequence can include, but is not limited to,prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and evensynthetic DNA sequences. If the coding sequence is intended forexpression in a eukaryotic cell, a polyadenylation signal andtranscription termination sequence will usually be located 3′ to thecoding sequence.

“Open reading frame” is abbreviated ORF and means a length of nucleicacid sequence, either DNA, cDNA or RNA, that comprises a translationstart signal or initiation codon, such as an ATG or AUG, and atermination codon and can be potentially translated into a polypeptidesequence.

The term “head-to-head” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-head orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 5′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds away from the 5′ end ofthe other polynucleotide. The term “head-to-head” may be abbreviated(5′)-to-(5′) and may also be indicated by the symbols (← →) or(3′←5′5′→3′).

The term “tail-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a tail-to-tail orientation when the 3′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds toward the otherpolynucleotide. The term “tail-to-tail” may be abbreviated (3′)-to-(3′)and may also be indicated by the symbols (→ ←) or (5′→3′3′←5′).

The term “head-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-tail orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds in the same directionas that of the other polynucleotide. The term “head-to-tail” may beabbreviated (5′)-to-(3′) and may also be indicated by the symbols (→ →)or (5′→3′5′→3′).

The term “downstream” refers to a nucleotide sequence that is located 3′to reference nucleotide sequence. In particular, downstream nucleotidesequences generally relate to sequences that follow the starting pointof transcription. For example, the translation initiation codon of agene is located downstream of the start site of transcription.

The term “upstream” refers to a nucleotide sequence that is located 5′to reference nucleotide sequence. In particular, upstream nucleotidesequences generally relate to sequences that are located on the 5′ sideof a coding sequence or starting point of transcription. For example,most promoters are located upstream of the start site of transcription.

The terms “restriction endonuclease” and “restriction enzyme” refer toan enzyme that binds and cuts within a specific nucleotide sequencewithin double stranded DNA.

“Homologous recombination” refers to the insertion of a foreign DNAsequence into another DNA molecule, e.g., insertion of a vector in achromosome. Preferably, the vector targets a specific chromosomal sitefor homologous recombination. For specific homologous recombination, thevector will contain sufficiently long regions of homology to sequencesof the chromosome to allow complementary binding and incorporation ofthe vector into the chromosome. Longer regions of homology, and greaterdegrees of sequence similarity, may increase the efficiency ofhomologous recombination.

Several methods known in the art may be used to propagate apolynucleotide according to the invention. Once a suitable host systemand growth conditions are established, recombinant expression vectorscan be propagated and prepared in quantity. As described herein, theexpression vectors which can be used include, but are not limited to,the following vectors or their derivatives: human or animal viruses suchas vaccinia virus or adenovirus; insect viruses such as baculovirus;yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid andcosmid DNA vectors, to name but a few.

A “vector” is any means for the cloning of and/or transfer of a nucleicacid into a host cell. A vector may be a replicon to which another DNAsegment may be attached so as to bring about the replication of theattached segment. A “replicon” is any genetic element (e.g., plasmid,phage, cosmid, chromosome, virus) that functions as an autonomous unitof DNA replication in vivo, i.e., capable of replication under its owncontrol. The term “vector” includes both viral and nonviral means forintroducing the nucleic acid into a cell in vitro, ex vivo or in vivo. Alarge number of vectors known in the art may be used to manipulatenucleic acids, incorporate response elements and promoters into genes,etc. Possible vectors include, for example, plasmids or modified virusesincluding, for example bacteriophages such as lambda derivatives, orplasmids such as pBR322 or pUC plasmid derivatives, or the Bluescriptvector. For example, the insertion of the DNA fragments corresponding toresponse elements and promoters into a suitable vector can beaccomplished by ligating the appropriate DNA fragments into a chosenvector that has complementary cohesive termini. Alternatively, the endsof the DNA molecules may be enzymatically modified or any site may beproduced by ligating nucleotide sequences (linkers) into the DNAtermini. Such vectors may be engineered to contain selectable markergenes that provide for the selection of cells that have incorporated themarker into the cellular genome. Such markers allow identificationand/or selection of host cells that incorporate and express the proteinsencoded by the marker.

Viral vectors, and particularly retroviral vectors, have been used in awide variety of gene delivery applications in cells, as well as livinganimal subjects. Viral vectors that can be used include but are notlimited to retrovirus, adeno-associated virus, pox, baculovirus,vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, andcaulimovirus vectors. Non-viral vectors include plasmids, liposomes,electrically charged lipids (cytofectins), DNA-protein complexes, andbiopolymers. In addition to a nucleic acid, a vector may also compriseone or more regulatory regions, and/or selectable markers useful inselecting, measuring, and monitoring nucleic acid transfer results(transfer to which tissues, duration of expression, etc.).

The term “plasmid” refers to an extra chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

A “cloning vector” is a “replicon”, which is a unit length of a nucleicacid, preferably DNA, that replicates sequentially and which comprisesan origin of replication, such as a plasmid, phage or cosmid, to whichanother nucleic acid segment may be attached so as to bring about thereplication of the attached segment. Cloning vectors may be capable ofreplication in one cell type and expression in another (“shuttlevector”).

Vectors may be introduced into the desired host cells by methods knownin the art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263: 14621-14624; and Hartmutet al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

A polynucleotide according to the invention can also be introduced invivo by lipofection. For the past decade, there has been increasing useof liposomes for encapsulation and transfection of nucleic acids invitro. Synthetic cationic lipids designed to limit the difficulties anddangers encountered with liposome-mediated transfection can be used toprepare liposomes for in vivo transfection of a gene encoding a marker(Felgner et al., 1987, PNAS 84:7413; Mackey, et al., 1988. Proc. Natl.Acad. Sci. U.S.A. 85:8027-8031; and Ulmer et al., 1993, Science259:1745-1748). The use of cationic lipids may promote encapsulation ofnegatively charged nucleic acids, and also promote fusion withnegatively charged cell membranes (Felgner and Ringold, 1989, Science337: 387-388). Particularly useful lipid compounds and compositions fortransfer of nucleic acids are described in International PatentPublications WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127.The use of lipofection to introduce exogenous genes into the specificorgans in vivo has certain practical advantages. Molecular targeting ofliposomes to specific cells represents one area of benefit. It is clearthat directing transfection to particular cell types would beparticularly preferred in a tissue with cellular heterogeneity, such aspancreas, liver, kidney, and the brain. Lipids may be chemically coupledto other molecules for the purpose of targeting (Mackey, et al., 1988,supra). Targeted peptides, e.g., hormones or neurotransmitters, andproteins such as antibodies, or non-peptide molecules could be coupledto liposomes chemically.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g.,WO95/21931), peptides derived from DNA binding proteins (e.g.,WO96/25508), or a cationic polymer (e.g., WO95/21931).

It is also possible to introduce a vector in vivo as a naked DNA plasmid(see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., 1992, Hum. Gene Ther. 3: 147-154; and Wu and Wu, 1987, J. Biol.Chem. 262: 4429-4432).

The term “transfection” means the uptake of exogenous or heterologousRNA or DNA by a cell. A cell has been “transfected” by exogenous orheterologous RNA or DNA when such RNA or DNA has been introduced insidethe cell. A cell has been “transformed” by exogenous or heterologous RNAor DNA when the transfected RNA or DNA effects a phenotypic change. Thetransforming RNA or DNA can be integrated (covalently linked) intochromosomal DNA making up the genome of the cell.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The term “genetic region” will refer to a region of a nucleic acidmolecule or a nucleotide sequence that comprises a gene encoding apolypeptide.

In addition, the recombinant vector comprising a polynucleotideaccording to the invention may include one or more origins forreplication in the cellular hosts in which their amplification or theirexpression is sought, markers or selectable markers.

The term “selectable marker” means an identifying factor, usually anantibiotic or chemical resistance gene, that is able to be selected forbased upon the marker gene's effect, i.e., resistance to an antibiotic,resistance to a herbicide, calorimetric markers, enzymes, fluorescentmarkers, and the like, wherein the effect is used to track theinheritance of a nucleic acid of interest and/or to identify a cell ororganism that has inherited the nucleic acid of interest. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl transferase gene, and the like.

The term “reporter gene” means a nucleic acid encoding an identifyingfactor that is able to be identified based upon the reporter gene'seffect, wherein the effect is used to track the inheritance of a nucleicacid of interest, to identify a cell or organism that has inherited thenucleic acid of interest, and/or to measure gene expression induction ortranscription. Examples of reporter genes known and used in the artinclude: luciferase (Luc), green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ),β-glucuronidase (Gus), and the like. Selectable marker genes may also beconsidered reporter genes.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. Promotersthat cause a gene to be expressed in a specific cell type are commonlyreferred to as “cell-specific promoters” or “tissue-specific promoters”.Promoters that cause a gene to be expressed at a specific stage ofdevelopment or cell differentiation are commonly referred to as“developmentally-specific promoters” or “cell differentiation-specificpromoters”. Promoters that are induced and cause a gene to be expressedfollowing exposure or treatment of the cell with an agent, biologicalmolecule, chemical, ligand, light, or the like that induces the promoterare commonly referred to as “inducible promoters” or “regulatablepromoters”. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined, DNAfragments of different lengths may have identical promoter activity.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced (if the coding sequence contains introns) and translated intothe protein encoded by the coding sequence.

“Transcriptional and translational control sequences” are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

The term “response element” means one or more cis-acting DNA elementswhich confer responsiveness on a promoter mediated through interactionwith the DNA-binding domains of the first chimeric gene. This DNAelement may be either palindromic (perfect or imperfect) in its sequenceor composed of sequence motifs or half sites separated by a variablenumber of nucleotides. The half sites can be similar or identical andarranged as either direct or inverted repeats or as a single half siteor multimers of adjacent half sites in tandem. The response element maycomprise a minimal promoter isolated from different organisms dependingupon the nature of the cell or organism into which the response elementwill be incorporated. The DNA binding domain of the first hybrid proteinbinds, in the presence or absence of a ligand, to the DNA sequence of aresponse element to initiate or suppress transcription of downstreamgene(s) under the regulation of this response element. Examples of DNAsequences for response elements of the natural ecdysone receptorinclude: RRGG/TTCANTGAC/ACYY (see Cherbas L., et. al., (1991), GenesDev. 5, 120-131); AGGTCAN_((n))AGGTCA, where N_((n)) can be one or morespacer nucleotides (see D'Avino P P., et. al., (1995), Mol. Cell.Endocrinol, 113, 1-9); and GGGTTGAATGAATT (see Antoniewski C., et. al.,(1994). Mol. Cell. Biol. 14, 4465-4474).

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from anucleic acid or polynucleotide. Expression may also refer to translationof mRNA into a protein or polypeptide.

The terms “cassette”, “expression cassette” and “gene expressioncassette” refer to a segment of DNA that can be inserted into a nucleicacid or polynucleotide at specific restriction sites or by homologousrecombination. The segment of DNA comprises a polynucleotide thatencodes a polypeptide of interest, and the cassette and restrictionsites are designed to ensure insertion of the cassette in the properreading frame for transcription and translation. “Transformationcassette” refers to a specific vector comprising a polynucleotide thatencodes a polypeptide of interest and having elements in addition to thepolynucleotide that facilitate transformation of a particular host cell.Cassettes, expression cassettes, gene expression cassettes andtransformation cassettes of the invention may also comprise elementsthat allow for enhanced expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

For purposes of this invention, the term “gene switch” refers to thecombination of a response element associated with a promoter, and an EcRbased system which in the presence of one or more ligands, modulates theexpression of a gene into which the response element and promoter areincorporated.

The terms “modulate” and “modulates” mean to induce, reduce or inhibitnucleic acid or gene expression, resulting in the respective induction,reduction or inhibition of protein or polypeptide production.

The plasmids or vectors according to the invention may further compriseat least one promoter suitable for driving expression of a gene in ahost cell. The term “expression vector” means a vector, plasmid orvehicle designed to enable the expression of an inserted nucleic acidsequence following transformation into the host. The cloned gene, i.e.,the inserted nucleic acid sequence, is usually placed under the controlof control elements such as a promoter, a minimal promoter, an enhancer,or the like. Initiation control regions or promoters, which are usefulto drive expression of a nucleic acid in the desired host cell arenumerous and familiar to those skilled in the art. Virtually anypromoter capable of driving these genes is suitable for the presentinvention including but not limited to: viral promoters, bacterialpromoters, animal promoters, mammalian promoters, synthetic promoters,constitutive promoters, tissue specific promoter, developmental specificpromoters, inducible promoters, light regulated promoters; CYC1, HIS3,GAL1, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO,TP1, alkaline phosphatase promoters (useful for expression inSaccharomyces); AOX1 promoter (useful for expression in Pichia);β-lactamase, lac, ara, tet, trp, IP_(L), IP_(R), T7, tac, and trcpromoters (useful for expression in Escherichia coli); light regulated-,seed specific-, pollen specific-, ovary specific-, pathogenesis ordisease related-, cauliflower mosaic virus 35S, CMV 35S minimal, cassavavein mosaic virus (CsVMV), chlorophyll a/b binding protein, ribulose1,5-bisphosphate carboxylase, shoot-specific, root specific, chitinase,stress inducible, rice tungro bacilliform virus, plant super-promoter,potato leucine aminopeptidase, nitrate reductase, mannopine synthase,nopaline synthase, ubiquitin, zein protein, and anthocyanin promoters(useful for expression in plant cells); animal and mammalian promotersknown in the art include, but are not limited to, the SV40 early (SV40e)promoter region, the promoter contained in the 3′ long terminal repeat(LTR) of Rous sarcoma virus (RSV), the promoters of the E1A or majorlate promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus(CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase(TK) promoter, a baculovirus IE1 promoter, an elongation factor 1 alpha(EF1) promoter, a phosphoglycerate kinase (PGK) promoter, a ubiquitin(Ubc) promoter, an albumin promoter, the regulatory sequences of themouse metallothionein-L promoter and transcriptional control regions,the ubiquitous promoters (HPRT, vimentin, α-actin, tubulin and thelike), the promoters of the intermediate filaments (desmin,neurofilaments, keratin, GFAP, and the like), the promoters oftherapeutic genes (of the MDR, CFTR or factor VIII type, and the like),pathogenesis or disease related-promoters, and promoters that exhibittissue specificity and have been utilized in transgenic animals, such asthe elastase I gene control region which is active in pancreatic acinarcells; insulin gene control region active in pancreatic beta cells,immunoglobulin gene control region active in lymphoid cells, mousemammary tumor virus control region active in testicular, breast,lymphoid and mast cells; albumin gene, Apo AI and Apo AII controlregions active in liver, alpha-fetoprotein gene control region active inliver, alpha 1-antitrypsin gene control region active in the liver,beta-globin gene control region active in myeloid cells, myelin basicprotein gene control region active in oligodendrocyte cells in thebrain, myosin light chain-2 gene control region active in skeletalmuscle, and gonadotropic releasing hormone gene control region active inthe hypothalamus, pyruvate kinase promoter, villin promoter, promoter ofthe fatty acid binding intestinal protein, promoter of the smooth musclecell α-actin, and the like. In addition, these expression sequences maybe modified by addition of enhancer or regulatory sequences and thelike.

Enhancers that may be used in embodiments of the invention include butare not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer,an elongation factor 1 (EFI) enhancer, yeast enhancers, viral geneenhancers, and the like.

Termination control regions, i.e., terminator or polyadenylationsequences, may also be derived from various genes native to thepreferred hosts. Optionally, a termination site may be unnecessary,however, it is most preferred if included. In a preferred embodiment ofthe invention, the termination control region may be comprise or bederived from a synthetic sequence, synthetic polyadenylation signal, anSV40 late polyadenylation signal, an SV40 polyadenylation signal, abovine growth hormone (BGH) polyadenylation signal, viral terminatorsequences, or the like.

The terms “3′non-coding sequences” or “3′ untranslated region (UTR)”refer to DNA sequences located downstream (3′) of a coding sequence andmay comprise polyadenylation [poly(A)] recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor.

“Regulatory region” means a nucleic acid sequence that regulates theexpression of a second nucleic acid sequence. A regulatory region mayinclude sequences which are naturally responsible for expressing aparticular nucleic acid (a homologous region) or may include sequencesof a different origin that are responsible for expressing differentproteins or even synthetic proteins (a heterologous region). Inparticular, the sequences can be sequences of prokaryotic, eukaryotic,or viral genes or derived sequences that stimulate or represstranscription of a gene in a specific or non-specific manner and in aninducible or non-inducible manner. Regulatory regions include origins ofreplication, RNA splice sites, promoters, enhancers, transcriptionaltermination sequences, and signal sequences which direct the polypeptideinto the secretory pathways of the target cell.

A regulatory region from a “heterologous source” is a regulatory regionthat is not naturally associated with the expressed nucleic acid.Included among the heterologous regulatory regions are regulatoryregions from a different species, regulatory regions from a differentgene, hybrid regulatory sequences, and regulatory sequences which do notoccur in nature, but which are designed by one having ordinary skill inthe art.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene. The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or thecoding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA,or other RNA that is not translated yet has an effect on cellularprocesses.

A “polypeptide” is a polymeric compound comprised of covalently linkedamino acid residues. Amino acids have the following general structure:

Amino acids are classified into seven groups on the basis of the sidechain R: (1) aliphatic side chains, (2) side chains containing ahydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup. A polypeptide of the invention preferably comprises at leastabout 14 amino acids.

A “protein” is a polypeptide that performs a structural or functionalrole in a living cell.

An “isolated polypeptide” or “isolated protein” is a polypeptide orprotein that is substantially free of those compounds that are normallyassociated therewith in its natural state (e.g., other proteins orpolypeptides, nucleic acids, carbohydrates, lipids). “Isolated” is notmeant to exclude artificial or synthetic mixtures with other compounds,or the presence of impurities which do not interfere with biologicalactivity, and which may be present, for example, due to incompletepurification, addition of stabilizers, or compounding into apharmaceutically acceptable preparation.

A “substitution mutant polypeptide” or a “substitution mutant” will beunderstood to mean a mutant polypeptide comprising a substitution of atleast one (1) wild-type or naturally occurring amino acid with adifferent amino acid relative to the wild-type or naturally occurringpolypeptide. A substitution mutant polypeptide may comprise only one (1)wild-type or naturally occurring amino acid substitution and may bereferred to as a “point mutant” or a “single point mutant” polypeptide.Alternatively, a substitution mutant polypeptide may comprise asubstitution of two (2) or more wild-type or naturally occurring aminoacids with 2 or more amino acids relative to the wild-type or naturallyoccurring polypeptide. According to the invention, a Group H nuclearreceptor ligand binding domain polypeptide comprising a substitutionmutation comprises a substitution of at least one (1) wild-type ornaturally occurring amino acid with a different amino acid relative tothe wild-type or naturally occurring Group H nuclear receptor ligandbinding domain polypeptide.

Wherein the substitution mutant polypeptide comprises a substitution oftwo (2) or more wild-type or naturally occurring amino acids, thissubstitution may comprise either an equivalent number of wild-type ornaturally occurring amino acids deleted for the substitution, i.e., 2wild-type or naturally occurring amino acids replaced with 2non-wild-type or non-naturally occurring amino acids, or anon-equivalent number of wild-type amino acids deleted for thesubstitution, i.e., 2 wild-type amino acids replaced with 1non-wild-type amino acid (a substitution+deletion mutation), or 2wild-type amino acids replaced with 3 non-wild-type amino acids (asubstitution+insertion mutation).

Substitution mutants may be described using an abbreviated nomenclaturesystem to indicate the amino acid residue and number replaced within thereference polypeptide sequence and the new substituted amino acidresidue. For example, a substitution mutant in which the twentieth(20^(th)) amino acid residue of a polypeptide is substituted may beabbreviated as “x20z”, wherein “x” is the amino acid to be replaced,“20” is the amino acid residue position or number within thepolypeptide, and “z” is the new substituted amino acid. Therefore, asubstitution mutant abbreviated interchangeably as “E20A” or “Glu20Ala”indicates that the mutant comprises an alanine residue (commonlyabbreviated in the art as “A” or “Ala”) in place of the glutamic acid(commonly abbreviated in the art as “E” or “Glu”) at position 20 of thepolypeptide.

A substitution mutation may be made by any technique for mutagenesisknown in the art, including but not limited to, in vitro site-directedmutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem. 253: 6551;Zoller and Smith, 1984, DNA 3: 479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83: 710),use of TAB® linkers (Pharmacia), restriction endonucleasedigestion/fragment deletion and substitution,PCR-mediated/oligonucleotide-directed mutagenesis, and the like.PCR-based techniques are preferred for site-directed mutagenesis (seeHiguchi, 1989, “Using PCR to Engineer DNA”, in PCR Technology:Principles and Applications for DNA Amplification, H. Erlich, ed.,Stockton Press, Chapter 6, pp. 61-70).

“Fragment” of a polypeptide according to the invention will beunderstood to mean a polypeptide whose amino acid sequence is shorterthan that of the reference polypeptide and which comprises, over theentire portion with these reference polypeptides, an identical aminoacid sequence. Such fragments may, where appropriate, be included in alarger polypeptide of which they are a part. Such fragments of apolypeptide according to the invention may have a length of at least 2,3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30,35, 40, 45, 50, 100, 200, 240, or 300 amino acids.

A “variant” of a polypeptide or protein is any analogue, fragment,derivative, or mutant which is derived from a polypeptide or protein andwhich retains at least one biological property of the polypeptide orprotein. Different variants of the polypeptide or protein may exist innature. These variants may be allelic variations characterized bydifferences in the nucleotide sequences of the structural gene codingfor the protein, or may involve differential splicing orpost-translational modification. The skilled artisan can producevariants having single or multiple amino acid substitutions, deletions,additions, or replacements. These variants may include, inter alia: (a)variants in which one or more amino acid residues are substituted withconservative or non-conservative amino acids, (b) variants in which oneor more amino acids are added to the polypeptide or protein, (c)variants in which one or more of the amino acids includes a substituentgroup, and (d) variants in which the polypeptide or protein is fusedwith another polypeptide such as serum albumin. The techniques forobtaining these variants, including genetic (suppressions, deletions,mutations, etc.), chemical, and enzymatic techniques, are known topersons having ordinary skill in the art. A variant polypeptidepreferably comprises at least about 14 amino acids.

A “heterologous protein” refers to a protein not naturally produced inthe cell.

A “mature protein” refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of mRNA; i.e., with pre-and propeptides still present. Pre- and propeptides may be but are notlimited to intracellular localization signals.

The term “signal peptide” refers to an amino terminal polypeptidepreceding the secreted mature protein. The signal peptide is cleavedfrom and is therefore not present in the mature protein. Signal peptideshave the function of directing and translocating secreted proteinsacross cell membranes. Signal peptide is also referred to as signalprotein.

A “signal sequence” is included at the beginning of the coding sequenceof a protein to be expressed on the surface of a cell. This sequenceencodes a signal peptide, N-terminal to the mature polypeptide, thatdirects the host cell to translocate the polypeptide. The term“translocation signal sequence” is used herein to refer to this sort ofsignal sequence. Translocation signal sequences can be found associatedwith a variety of proteins native to eukaryotes and prokaryotes, and areoften functional in both types of organisms.

The term “homology” refers to the percent of identity between twopolynucleotide or two polypeptide moieties. The correspondence betweenthe sequence from one moiety to another can be determined by techniquesknown to the art. For example, homology can be determined by a directcomparison of the sequence information between two polypeptide moleculesby aligning the sequence information and using readily availablecomputer programs. Alternatively, homology can be determined byhybridization of polynucleotides under conditions that form stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s) and size determination of thedigested fragments.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., 1987, Cell 50:667.). Such proteins (and their encoding genes)have sequence homology, as reflected by their high degree of sequencesimilarity. However, in common usage and in the instant application, theterm “homologous,” when modified with an adverb such as “highly,” mayrefer to sequence similarity and not a common evolutionary origin.

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that may or may not share a commonevolutionary origin (see Reeck et al., 1987, Cell 50: 667).

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 50%(preferably at least about 75%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Sambrook et al., 1989, supra.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the protein encoded by the DNA sequence. “Substantially similar” alsorefers to nucleic acid fragments wherein changes in one or morenucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotide bases that donot substantially affect the functional properties of the resultingtranscript. It is therefore understood that the invention encompassesmore than the specific exemplary sequences. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts.

Moreover, the skilled artisan recognizes that substantially similarsequences encompassed by this invention are also defined by theirability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65°C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS), withthe sequences exemplified herein. Substantially similar nucleic acidfragments of the instant invention are those nucleic acid fragmentswhose DNA sequences are at least 70% identical to the DNA sequence ofthe nucleic acid fragments reported herein. Preferred substantiallynucleic acid fragments of the instant invention are those nucleic acidfragments whose DNA sequences are at least 80% identical to the DNAsequence of the nucleic acid fragments reported herein. More preferrednucleic acid fragments are at least 90% identical to the DNA sequence ofthe nucleic acid fragments reported herein. Even more preferred arenucleic acid fragments that are at least 95% identical to the DNAsequence of the nucleic acid fragments reported herein.

Two amino acid sequences are “substantially homologous” or“substantially similar” when greater than about 40% of the amino acidsare identical, or greater than 60% are similar (functionally identical).Preferably, the similar or homologous sequences are identified byalignment using, for example, the GCG (Genetics Computer Group, ProgramManual for the GCG Package, Version 7, Madison, Wis.) pileup program.

The term “corresponding to” is used herein to refer to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. A nucleic acid or amino acid sequence alignment may includespaces. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

A “substantial portion” of an amino acid or nucleotide sequencecomprises enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to putatively identify that polypeptide orgene, either by manual evaluation of the sequence by one skilled in theart, or by computer-automated sequence comparison and identificationusing algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul, S. F., et al., (1993) J. Mol. Biol. 215: 403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M.and Devereux, J., eds.) Stockton Press, New York (1991). Preferredmethods to determine identity are designed to give the best matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequencesmay be performed using the Clustal method of alignment (Higgins andSharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method may be selected: KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include but is not limited to the GCG suite of programs (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.),BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410(1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715USA). Within the context of this application it will be understood thatwhere sequence analysis software is used for analysis, that the resultsof the analysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters which originally load with thesoftware when first initialized.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments that are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

As used herein, two or more individually operable gene regulationsystems are said to be “orthogonal” when; a) modulation of each of thegiven systems by its respective ligand, at a chosen concentration,results in a measurable change in the magnitude of expression of thegene of that system, and b) the change is statistically significantlydifferent than the change in expression of all other systemssimultaneously operable in the cell, tissue, or organism, regardless ofthe simultaneity or sequentially of the actual modulation. Preferably,modulation of each individually operable gene regulation system effectsa change in gene expression at least 2-fold greater than all otheroperable systems in the cell, tissue, or organism. More preferably, thechange is at least 5-fold greater. Even more preferably, the change isat least 10-fold greater. Still more preferably, the change is at least100 fold greater. Even still more preferably, the change is at least500-fold greater. Ideally, modulation of each of the given systems byits respective ligand at a chosen concentration results in a measurablechange in the magnitude of expression of the gene of that system and nomeasurable change in expression of all other systems operable in thecell, tissue, or organism. In such cases the multiple inducible generegulation system is said to be “fully orthogonal”. The presentinvention is useful to search for orthogonal ligands and orthogonalreceptor-based gene expression systems such as those described inco-pending U.S. application Ser. No. 09/965,697, which is incorporatedherein by reference in its entirety.

The term “modulate” means the ability of a given ligand/receptor complexto induce or suppress the transactivation of an exogenous gene.

The term “exogenous gene” means a gene foreign to the subject, that is,a gene which is introduced into the subject through a transformationprocess, an unmutated version of an endogenous mutated gene or a mutatedversion of an endogenous unmutated gene. The method of transformation isnot critical to this invention and may be any method suitable for thesubject known to those in the art. For example, transgenic plants areobtained by regeneration from the transformed cells. Numeroustransformation procedures are known from the literature such asagroinfection using Agrobacterium tumefaciens or its T₁ plasmid,electroporation, microinjection of plant cells and protoplasts, andmicroprojectile transformation. Complementary techniques are known fortransformation of animal cells and regeneration of such transformedcells in transgenic animals. Exogenous genes can be either natural orsynthetic genes and therapeutic genes which are introduced into thesubject in the form of DNA or RNA which may function through a DNAintermediate such as by reverse transcriptase. Such genes can beintroduced into target cells, directly introduced into the subject, orindirectly introduced by the transfer of transformed cells into thesubject. The term “therapeutic gene” means a gene which imparts abeneficial function to the host cell in which such gene is expressed.Therapeutic genes are not naturally found in host cells.

The term “ecdysone receptor complex” generally refers to a heterodimericprotein complex consisting of two members of the steroid receptorfamily, ecdysone receptor (“EcR”) and ultraspiracle (“USP”) proteins(see Yao, T. P., et. al. (1993) Nature 366, 476-479; Yao, T.-P., et.al., (1992) Cell 71, 63-72). The functional ecdysteroid receptor complexmay also include additional protein(s) such as immunophilins. Additionalmembers of the steroid receptor family of proteins, known astranscriptional factors (such as DHR38, betaFTZ-1 or other insecthomologs), may also be ligand dependent or independent partners for EcRand/or USP. The ecdysone receptor complex can also be a heterodimer ofecdysone receptor protein and the vertebrate homolog of ultraspiracleprotein, retinoic acid-X-receptor (“RXR”) protein. Homodimer complexesof the ecdysone receptor protein or USP may also be functional undersome circumstances.

An ecdysteroid receptor complex can be activated by an activeecdysteroid or non-steroidal ligand bound to one of the proteins of thecomplex, inclusive of EcR, but not excluding other proteins of thecomplex.

The ecdysone receptor complex includes proteins which are members of thesteroid receptor superfamily wherein all members are characterized bythe presence of an amino-terminal transactivation domain, a DNA bindingdomain (“DBD”), and a ligand binding domain (“LBD”) separated by a hingeregion. Some members of the family may also have another transactivationdomain on the carboxy-terminal side of the LBD. The DBD is characterizedby the presence of two cysteine zinc fingers between which are two aminoacid motifs, the P-box and the D-box, which confer specificity forecdysone response elements. These domains may be either native,modified, or chimeras of different domains of heterologous receptorproteins.

The DNA sequences making up the exogenous gene, the response element,and the ecdysone receptor complex may be incorporated intoarchaebacteria, procaryotic cells such as Escherichia coli, Bacillussubtilis, or other enterobacteria, or eucaryotic cells such as plant oranimal cells. However, because many of the proteins expressed by thegene are processed incorrectly in bacteria, eucaryotic cells arepreferred. The cells may be in the form of single cells or multicellularorganisms. The nucleotide sequences for the exogenous gene, the responseelement, and the receptor complex can also be incorporated as RNAmolecules, preferably in the form of functional viral RNAs such astobacco mosaic virus. Of the eucaryotic cells, vertebrate cells arepreferred because they naturally lack the molecules which conferresponses to the ligands of this invention for the ecdysone receptor. Asa result, they are insensitive to the ligands of this invention. Thus,the ligands of this invention will have negligible physiological orother effects on transformed cells, or the whole organism. Therefore,cells can grow and express the desired product, substantially unaffectedby the presence of the ligand itself.

The term “subject” means an intact plant or animal or a cell from aplant or animal. It is also anticipated that the ligands will workequally well when the subject is a fungus or yeast. When the subject isan intact animal, preferably the animal is a vertebrate, most preferablya mammal.

The ligands of the present invention, when used with the ecdysonereceptor complex which in turn is bound to the response element linkedto an exogenous gene, provide the means for external temporal regulationof expression of the exogenous gene. The order in which the variouscomponents bind to each other, that is, ligand to receptor complex andreceptor complex to response element, is not critical. Typically,modulation of expression of the exogenous gene is in response to thebinding of the ecdysone receptor complex to a specific control, orregulatory, DNA element. The ecdysone receptor protein, like othermembers of the steroid receptor family, possesses at least threedomains, a transactivation domain, a DNA binding domain, and a ligandbinding domain. This receptor, like a subset of the steroid receptorfamily, also possesses less well-defined regions responsible forheterodimerization properties. Binding of the ligand to the ligandbinding domain of ecdysone receptor protein, after heterodimerizationwith USP or RXR protein, enables the DNA binding domains of theheterodimeric proteins to bind to the response element in an activatedform, thus resulting in expression or suppression of the exogenous gene.This mechanism does not exclude the potential for ligand binding toeither EcR or USP, and the resulting formation of active homodimercomplexes (e.g. EcR+EcR or USP+USP). Preferably, one or more of thereceptor domains can be varied producing a chimeric gene switch.Typically, one or more of the three domains may be chosen from a sourcedifferent than the source of the other domains so that the chimericreceptor is optimized in the chosen host cell or organism fortransactivating activity, complementary binding of the ligand, andrecognition of a specific response element. In addition, the responseelement itself can be modified or substituted with response elements forother DNA binding protein domains such as the GAL-4 protein from yeast(see Sadowski, et. al. (1988) Nature, 335, 563-564) or LexA protein fromE. coli (see Brent and Ptashne (1985), Cell, 43, 729-736) to accommodatechimeric ecdysone receptor complexes. Another advantage of chimericsystems is that they allow choice of a promoter used to drive theexogenous gene according to a desired end result. Such double controlcan be particularly important in areas of gene therapy, especially whencytotoxic proteins are produced, because both the timing of expressionas well as the cells wherein expression occurs can be controlled. Theterm “promoter” means a specific nucleotide sequence recognized by RNApolymerase. The sequence is the site at which transcription can bespecifically initiated under proper conditions. When exogenous genes,operatively linked to a suitable promoter, are introduced into the cellsof the subject, expression of the exogenous genes is controlled by thepresence of the ligand of this invention. Promoters may beconstitutively or inducibly regulated or may be tissue-specific (thatis, expressed only in a particular type of cell) or specific to certaindevelopmental stages of the organism.

Another aspect of this invention is a method to modulate the expressionof one or more exogenous genes in a subject, comprising administering tothe subject an effective amount, that is, the amount required to elicitthe desired gene expression or suppression, of a ligand comprising acompound of the present invention and wherein the cells of the subjectcontain:

-   -   a) an ecdysone receptor complex comprising:        -   1) a DNA binding domain;        -   2) a binding domain for the ligand; and        -   3) a transactivation domain; and    -   b) a DNA construct comprising:        -   1) the exogenous gene; and        -   2) a response element;            wherein the exogenous gene is under the control of the            response element; and binding of the DNA binding domain to            the response element in the presence of the ligand results            in activation or suppression of the gene.

A related aspect of this invention is a method for regulating endogenousor heterologous gene expression in a transgenic subject comprisingcontacting a ligand comprising a compound of the present invention withan ecdysone receptor within the cells of the subject wherein the cellscontain a DNA binding sequence for the ecdysone receptor and whereinformation of an ecdysone receptor-ligand-DNA binding sequence complexinduces expression of the gene.

Another aspect of the present invention is a method for producing apolypeptide comprising the steps of:

-   -   a) selecting a cell which is substantially insensitive to        exposure to a ligand comprising a compound of the present        invention;    -   b) introducing into the cell:        -   1) a DNA construct comprising:            -   i) an exogenous gene encoding the polypeptide; and            -   ii) a response element;    -   wherein the gene is under the control of the response element;        and        -   2) an ecdysone receptor complex comprising:            -   i) a DNA binding domain;            -   ii) a binding domain for the ligand; and            -   iii) a transactivation domain; and    -   c) exposing the cell to the ligand.

As well as the advantage of temporally controlling polypeptideproduction by the cell, this aspect of the invention provides a furtheradvantage, in those cases when accumulation of such a polypeptide candamage the cell, in that expression of the polypeptide may be limited toshort periods. Such control is particularly important when the exogenousgene is a therapeutic gene. Therapeutic genes may be called upon toproduce polypeptides which control needed functions, such as theproduction of insulin in diabetic patients. They may also be used toproduce damaging or even lethal proteins, such as those lethal to cancercells. Such control may also be important when the protein levelsproduced may constitute a metabolic drain on growth or reproduction,such as in transgenic plants.

Numerous genomic and cDNA nucleic acid sequences coding for a variety ofpolypeptides are well known in the art. Exogenous genetic materialuseful with the ligands of this invention include genes that encodebiologically active proteins of interest, such as, for example,secretory proteins that can be released from a cell; enzymes that canmetabolize a substrate from a toxic substance to a non-toxic substance,or from an inactive substance to an active substance; regulatoryproteins; cell surface receptors; and the like. Useful genes alsoinclude genes that encode blood clotting factors, hormones such asinsulin, parathyroid hormone, luteinizing hormone releasing factor,alpha and beta seminal inhibins, and human growth hormone; genes thatencode proteins such as enzymes, the absence of which leads to theoccurrence of an abnormal state; genes encoding cytokines or lymphokinessuch as interferons, granulocytic macrophage colony stimulating factor,colony stimulating factor-1, tumor necrosis factor, and erythropoietin;genes encoding inhibitor substances such as alpha₁-antitrypsin, genesencoding substances that function as drugs such as diphtheria andcholera toxins; and the like. Useful genes also include those useful forcancer therapies and to treat genetic disorders. Those skilled in theart have access to nucleic acid sequence information for virtually allknown genes and can either obtain the nucleic acid molecule directlyfrom a public depository, the institution that published the sequence,or employ routine methods to prepare the molecule.

For gene therapy use, the ligands described herein may be taken up inpharmaceutically acceptable carriers, such as, for example, solutions,suspensions, tablets, capsules, ointments, elixirs, and injectablecompositions. Pharmaceutical preparations may contain from 0.01% to 99%by weight of the ligand. Preparations may be either in single ormultiple dose forms. The amount of ligand in any particularpharmaceutical preparation will depend upon the effective dose, that is,the dose required to elicit the desired gene expression or suppression.

Suitable routes of administering the pharmaceutical preparations includeoral, rectal, topical (including dermal, buccal and sublingual),vaginal, parenteral (including subcutaneous, intramuscular, intravenous,intradermal, intrathecal and epidural) and by naso-gastric tube. It willbe understood by those skilled in the art that the preferred route ofadministration will depend upon the condition being treated and may varywith factors such as the condition of the recipient.

The ligands described herein may also be administered in conjunctionwith other pharmaceutically active compounds. It will be understood bythose skilled in the art that pharmaceutically active compounds to beused in combination with the ligands described herein will be selectedin order to avoid adverse effects on the recipient or undesirableinteractions between the compounds. Examples of other pharmaceuticallyactive compounds which may be used in combination with the ligandsinclude, for example, AIDS chemotherapeutic agents, amino acidderivatives, analgesics, anesthetics, anorectal products, antacids andantiflatulents, antibiotics, anticoagulants, antidotes, antifibrinolyticagents, antihistamines, anti-inflamatory agents, antineoplastics,antiparasitics, antiprotozoals, antipyretics, antiseptics,antispasmodics and anticholinergics, antivirals, appetite suppressants,arthritis medications, biological response modifiers, bone metabolismregulators, bowel evacuants, cardiovascular agents, central nervoussystem stimulants, cerebral metabolic enhancers, cerumenolytics,cholinesterase inhibitors, cold and cough preparations, colonystimulating factors, contraceptives, cytoprotective agents, dentalpreparations, deodorants, dermatologicals, detoxifying agents, diabetesagents, diagnostics, diarrhea medications, dopamine receptor agonists,electrolytes, enzymes and digestants, ergot preparations, fertilityagents, fiber supplements, antifungal agents, galactorrhea inhibitors,gastric acid secretion inhibitors, gastrointestinal prokinetic agents,gonadotropin inhibitors, hair growth stimulants, hematinics,hemorrheologic agents, hemostatics, histamine H₂ receptor antagonists,hormones, hyperglycemic agents, hypolipidemics, immunosuppressants,laxatives, leprostatics, leukapheresis adjuncts, lung surfactants,migraine preparations, mucolytics, muscle relaxant antagonists, musclerelaxants, narcotic antagonists, nasal sprays, nausea medicationsnucleoside analogues, nutritional supplements, osteoporosispreparations, oxytocics, parasympatholytics, parasympathomimetics,Parkinsonism drugs, Penicillin adjuvants, phospholipids, plateletinhibitors, porphyria agents, prostaglandin analogues, prostaglandins,proton pump inhibitors, pruritus medications psychotropics, quinolones,respiratory stimulants, saliva stimulants, salt substitutes, sclerosingagents, skin wound preparations, smoking cessation aids, sulfonamides,sympatholytics, thrombolytics, Tourette's syndrome agents, tremorpreparations, tuberculosis preparations, uricosuric agents, urinarytract agents, uterine contractants, uterine relaxants, vaginalpreparations, vertigo agents, vitamin D analogs, vitamins, and medicalimaging contrast media. In some cases the ligands may be useful as anadjunct to drug therapy, for example, to “turn off” a gene that producesan enzyme that metabolizes a particular drug.

For agricultural applications, in addition to the applications describedabove, the ligands of this invention may also be used to control theexpression of pesticidal proteins such as Bacillus thuringiensis (Bt)toxin. Such expression may be tissue or plant specific. In addition,particularly when control of plant pests is also needed, one or morepesticides may be combined with the ligands described herein, therebyproviding additional advantages and effectiveness, including fewer totalapplications, than if the pesticides are applied separately. Whenmixtures with pesticides are employed, the relative proportions of eachcomponent in the composition will depend upon the relative efficacy andthe desired application rate of each pesticide with respect to thecrops, pests, and/or weeds to be treated. Those skilled in the art willrecognize that mixtures of pesticides may provide advantages such as abroader spectrum of activity than one pesticide used alone. Examples ofpesticides which can be combined in compositions with the ligandsdescribed herein include fungicides, herbicides, insecticides,miticides, and microbicides.

The ligands described herein can be applied to plant foliage as aqueoussprays by methods commonly employed, such as conventional high-literhydraulic sprays, low-liter sprays, air-blast, and aerial sprays. Thedilution and rate of application will depend upon the type of equipmentemployed, the method and frequency of application desired, and theligand application rate. It may be desirable to include additionaladjuvants in the spray tank. Such adjuvants include surfactants,dispersants, spreaders, stickers, antifoam agents, emulsifiers, andother similar materials described in McCutcheon's Emulsifiers andDetergents, McCutcheon's Emulsifiers and Detergents/FunctionalMaterials, and McCutcheon's Functional Materials, all published annuallyby McCutcheon Division of MC Publishing Company (New Jersey). Theligands can also be mixed with fertilizers or fertilizing materialsbefore their application. The ligands and solid fertilizing material canalso be admixed in mixing or blending equipment, or they can beincorporated with fertilizers in granular formulations. Any relativeproportion of fertilizer can be used which is suitable for the crops andweeds to be treated. The ligands described herein will commonly comprisefrom 5% to 50% of the fertilizing composition. These compositionsprovide fertilizing materials which promote the rapid growth of desiredplants, and at the same time control gene expression.

Host Cells and Non-Human Organisms of the Invention

As described above, ligands for modulating gene expression system of thepresent invention may be used to modulate gene expression in a hostcell. Expression in transgenic host cells may be useful for theexpression of various genes of interest. The present invention providesligands for modulation of gene expression in prokaryotic and eukaryotichost cells. Expression in transgenic host cells is useful for theexpression of various polypeptides of interest including but not limitedto antigens produced in plants as vaccines, enzymes like alpha-amylase,phytase, glucanes, and xylanse, genes for resistance against insects,nematodes, fungi, bacteria, viruses, and abiotic stresses, antigens,nutraceuticals, pharmaceuticals, vitamins, genes for modifying aminoacid content, herbicide resistance, cold, drought, and heat tolerance,industrial products, oils, protein, carbohydrates, antioxidants, malesterile plants, flowers, fuels, other output traits, therapeuticpolypeptides, pathway intermediates; for the modulation of pathwaysalready existing in the host for the synthesis of new productsheretofore not possible using the host; cell based assays; functionalgenomics assays, biotherapeutic protein production, proteomics assays,and the like. Additionally the gene products may be useful forconferring higher growth yields of the host or for enabling analternative growth mode to be utilized.

Thus, the present invention provides ligands for modulating geneexpression in an isolated host cell according to the invention. The hostcell may be a bacterial cell, a fungal cell, a nematode cell, an insectcell, a fish cell, a plant cell, an avian cell, an animal cell, or amammalian cell. In still another embodiment, the invention relates toligands for modulating gene expression in an host cell, wherein themethod comprises culturing the host cell as described above in culturemedium under conditions permitting expression of a polynucleotideencoding the nuclear receptor ligand binding domain comprising asubstitution mutation, and isolating the nuclear receptor ligand bindingdomain comprising a substitution mutation from the culture.

In a specific embodiment, the isolated host cell is a prokaryotic hostcell or a eukaryotic host cell. In another specific embodiment, theisolated host cell is an invertebrate host cell or a vertebrate hostcell. Preferably, the host cell is selected from the group consisting ofa bacterial cell, a fungal cell, a yeast cell, a nematode cell, aninsect cell, a fish cell, a plant cell, an avian cell, an animal cell,and a mammalian cell. More preferably, the host cell is a yeast cell, anematode cell, an insect cell, a plant cell, a zebrafish cell, a chickencell, a hamster cell, a mouse cell, a rat cell, a rabbit cell, a catcell, a dog cell, a bovine cell, a goat cell, a cow cell, a pig cell, ahorse cell, a sheep cell, a simian cell, a monkey cell, a chimpanzeecell, or a human cell. Examples of preferred host cells include, but arenot limited to, fungal or yeast species such as Aspergillus,Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, or bacterialspecies such as those in the genera Synechocystis, Synechococcus,Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces,Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcaligenes,Synechocystis, Anabaena, Thiobacillus, Methanobacterium and Klebsiella;plant species selected from the group consisting of an apple,Arabidopsis, bajra, banana, barley, beans, beet, blackgram, chickpea,chili, cucumber, eggplant, favabean, maize, melon, millet, mungbean,oat, okra, Panicum, papaya, peanut, pea, pepper, pigeonpea, pineapple,Phaseolus, potato, pumpkin, rice, sorghum, soybean, squash, sugarcane,sugarbeet, sunflower, sweet potato, tea, tomato, tobacco, watermelon,and wheat; animal; and mammalian host cells.

In a specific embodiment, the host cell is a yeast cell selected fromthe group consisting of a Saccharomyces, a Pichia, and a Candida hostcell.

In another specific embodiment, the host cell is a Caenorhabdus elegansnematode cell.

In another specific embodiment, the host cell is an insect cell.

In another specific embodiment, the host cell is a plant cell selectedfrom the group consisting of an apple, Arabidopsis, bajra, banana,barley, beans, beet, blackgram, chickpea, chili, cucumber, eggplant,favabean, maize, melon, millet, mungbean, oat, okra, Panicum, papaya,peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato, pumpkin,rice, sorghum, soybean, squash, sugarcane, sugarbeet, sunflower, sweetpotato, tea, tomato, tobacco, watermelon, and wheat cell.

In another specific embodiment, the host cell is a zebrafish cell.

In another specific embodiment, the host cell is a chicken cell.

In another specific embodiment, the host cell is a mammalian cellselected from the group consisting of a hamster cell, a mouse cell, arat cell, a rabbit cell, a cat cell, a dog cell, a bovine cell, a goatcell, a cow cell, a pig cell, a horse cell, a sheep cell, a monkey cell,a chimpanzee cell, and a human cell.

Host cell transformation is well known in the art and may be achieved bya variety of methods including but not limited to electroporation, viralinfection, plasmid/vector transfection, non-viral vector mediatedtransfection, Agrobacterium-mediated transformation, particlebombardment, and the like. Expression of desired gene products involvesculturing the transformed host cells under suitable conditions andinducing expression of the transformed gene. Culture conditions and geneexpression protocols in prokaryotic and eukaryotic cells are well knownin the art (see General Methods section of Examples). Cells may beharvested and the gene products isolated according to protocols specificfor the gene product.

In addition, a host cell may be chosen which modulates the expression ofthe inserted polynucleotide, or modifies and processes the polypeptideproduct in the specific fashion desired. Different host cells havecharacteristic and specific mechanisms for the translational andpost-translational processing and modification [e.g., glycosylation,cleavage (e.g., of signal sequence)] of proteins. Appropriate cell linesor host systems can be chosen to ensure the desired modification andprocessing of the foreign protein expressed. For example, expression ina bacterial system can be used to produce a non-glycosylated coreprotein product. However, a polypeptide expressed in bacteria may not beproperly folded. Expression in yeast can produce a glycosylated product.Expression in eukaryotic cells can increase the likelihood of “native”glycosylation and folding of a heterologous protein. Moreover,expression in mammalian cells can provide a tool for reconstituting, orconstituting, the polypeptide's activity. Furthermore, differentvector/host expression systems may affect processing reactions, such asproteolytic cleavages, to a different extent. The present invention alsorelates to a non-human organism comprising an isolated host cellaccording to the invention. In a specific embodiment, the non-humanorganism is a prokaryotic organism or a eukaryotic organism. In anotherspecific embodiment, the non-human organism is an invertebrate organismor a vertebrate organism.

Preferably, the non-human organism is selected from the group consistingof a bacterium, a fungus, a yeast, a nematode, an insect, a fish, aplant, a bird, an animal, and a mammal. More preferably, the non-humanorganism is a yeast, a nematode, an insect, a plant, a zebrafish, achicken, a hamster, a mouse, a rat, a rabbit, a cat, a dog, a bovine, agoat, a cow, a pig, a horse, a sheep, a simian, a monkey, or achimpanzee.

In a specific embodiment, the non-human organism is a yeast selectedfrom the group consisting of Saccharomyces, Pichia, and Candida.

In another specific embodiment, the non-human organism is a Caenorhabduselegans nematode.

In another specific embodiment, the non-human organism is a plantselected from the group consisting of an apple, Arabidopsis, bajra,banana, barley, beans, beet, blackgram, chickpea, chili, cucumber,eggplant, favabean, maize, melon, millet, mungbean, oat, okra, Panicum,papaya, peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato,pumpkin, rice, sorghum, soybean, squash, sugarcane, sugarbeet,sunflower, sweet potato, tea, tomato, tobacco, watermelon, and wheat.

In another specific embodiment, the non-human organism is a Mus musculusmouse.

Gene Expression Modulation System of the Invention

The present invention relates to a group of ligands that are useful inan ecdysone receptor-based inducible gene expression system. Aspresented herein, a novel group of ligands provides an improvedinducible gene expression system in both prokaryotic and eukaryotic hostcells. Thus, the present invention relates to ligands that are useful tomodulate expression of genes. In particular, the present inventionrelates to ligands having the ability to transactivate a gene expressionmodulation system comprising at least one gene expression cassette thatis capable of being expressed in a host cell comprising a polynucleotidethat encodes a polypeptide comprising a Group H nuclear receptor ligandbinding domain. Preferably, the Group H nuclear receptor ligand bindingis from an ecdysone receptor, a ubiquitous receptor, an orphan receptor1, a NER-1, a steroid hormone nuclear receptor 1, a retinoid X receptorinteracting protein-15, a liver X receptor β, a steroid hormone receptorlike protein, a liver X receptor, a liver X receptor α, a farnesoid Xreceptor, a receptor interacting protein 14, and a farnesol receptor.More preferably, the Group H nuclear receptor ligand binding domain isfrom an ecdysone receptor.

In a specific embodiment, the gene expression modulation systemcomprises a gene expression cassette comprising a polynucleotide thatencodes a polypeptide comprising a transactivation domain, a DNA-bindingdomain that recognizes a response element associated with a gene whoseexpression is to be modulated; and a Group H nuclear receptor ligandbinding domain comprising a substitution mutation. The gene expressionmodulation system may further comprise a second gene expression cassettecomprising: i) a response element recognized by the DNA-binding domainof the encoded polypeptide of the first gene expression cassette; ii) apromoter that is activated by the transactivation domain of the encodedpolypeptide of the first gene expression cassette; and iii) a gene whoseexpression is to be modulated.

In another specific embodiment, the gene expression modulation systemcomprises a gene expression cassette comprising a) a polynucleotide thatencodes a polypeptide comprising a transactivation domain, a DNA-bindingdomain that recognizes a response element associated with a gene whoseexpression is to be modulated; and a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, and b) a secondnuclear receptor ligand binding domain selected from the groupconsisting of a vertebrate retinoid X receptor ligand binding domain, aninvertebrate retinoid X receptor ligand binding domain, an ultraspiracleprotein ligand binding domain, and a chimeric ligand binding domaincomprising two polypeptide fragments, wherein the first polypeptidefragment is from a vertebrate retinoid X receptor ligand binding domain,an invertebrate retinoid X receptor ligand binding domain, or anultraspiracle protein ligand binding domain, and the second polypeptidefragment is from a different vertebrate retinoid X receptor ligandbinding domain, invertebrate retinoid X receptor ligand binding domain,or ultraspiracle protein ligand binding domain. The gene expressionmodulation system may further comprise a second gene expression cassettecomprising: i) a response element recognized by the DNA-binding domainof the encoded polypeptide of the first gene expression cassette; ii) apromoter that is activated by the transactivation domain of the encodedpolypeptide of the first gene expression cassette; and iii) a gene whoseexpression is to be modulated.

In another specific embodiment, the gene expression modulation systemcomprises a first gene expression cassette comprising a polynucleotidethat encodes a first polypeptide comprising a DNA-binding domain thatrecognizes a response element associated with a gene whose expression isto be modulated and a nuclear receptor ligand binding domain, and asecond gene expression cassette comprising a polynucleotide that encodesa second polypeptide comprising a transactivation domain and a nuclearreceptor ligand binding domain, wherein one of the nuclear receptorligand binding domains is a Group H nuclear receptor ligand bindingdomain comprising a substitution mutation. In a preferred embodiment,the first polypeptide is substantially free of a transactivation domainand the second polypeptide is substantially free of a DNA bindingdomain. For purposes of the invention, “substantially free” means thatthe protein in question does not contain a sufficient sequence of thedomain in question to provide activation or binding activity. The geneexpression modulation system may further comprise a third geneexpression cassette comprising: i) a response element recognized by theDNA-binding domain of the first polypeptide of the first gene expressioncassette; ii) a promoter that is activated by the transactivation domainof the second polypeptide of the second gene expression cassette; andiii) a gene whose expression is to be modulated.

Wherein when only one nuclear receptor ligand binding domain is a GroupH ligand binding domain comprising a substitution mutation, the othernuclear receptor ligand binding domain may be from any other nuclearreceptor that forms a dimer with the Group H ligand binding domaincomprising the substitution mutation. For example, when the Group Hnuclear receptor ligand binding domain comprising a substitutionmutation is an ecdysone receptor ligand binding domain comprising asubstitution mutation, the other nuclear receptor ligand binding domain(“partner”) may be from an ecdysone receptor, a vertebrate retinoid Xreceptor (RXR), an invertebrate RXR, an ultraspiracle protein (USP), ora chimeric nuclear receptor comprising at least two different nuclearreceptor ligand binding domain polypeptide fragments selected from thegroup consisting of a vertebrate RXR, an invertebrate RXR, and a USP(see co-pending applications PCT/US01/09050, PCT/US02/05235, andPCT/US02/05706, incorporated herein by reference in their entirety). The“partner” nuclear receptor ligand binding domain may further comprise atruncation mutation, a deletion mutation, a substitution mutation, oranother modification.

Preferably, the vertebrate RXR ligand binding domain is from a humanHomo sapiens, mouse Mus musculus, rat Rattus norvegicus, chicken Gallusgallus, pig Sus scrofa domestica, frog Xenopus laevis, zebrafish Daniorerio, tunicate Polyandrocarpa misakiensis, or jellyfish Tripedaliacysophora RXR.

Preferably, the invertebrate RXR ligand binding domain is from a locustLocusta migratoria ultraspiracle polypeptide (“LmUSP”), an ixodid tickAmblyomma americanum RXR homolog 1 (“AmaRXR1”), a ixodid tick Amblyommaamericanum RXR homolog 2 (“AmaRXR2”), a fiddler crab Celuca pugilatorRXR homolog (“CpRXR”), a beetle Tenebrio molitor RXR homolog (“TmRXR”),a honeybee Apis mellifera RXR homolog (“AmRXR”), an aphid Myzus persicaeRXR homolog (“MpRXR”), or a non-Dipteran/non-Lepidopteran RXR homolog.

Preferably, the chimeric RXR ligand binding domain comprises at leasttwo polypeptide fragments selected from the group consisting of avertebrate species RXR polypeptide fragment, an invertebrate species RXRpolypeptide fragment, and a non-Dipteran/non-Lepidopteran invertebratespecies RXR homolog polypeptide fragment. A chimeric RXR ligand bindingdomain for use in the present invention may comprise at least twodifferent species RXR polypeptide fragments, or when the species is thesame, the two or more polypeptide fragments may be from two or moredifferent isoforms of the species RXR polypeptide fragment.

In a preferred embodiment, the chimeric RXR ligand binding domaincomprises at least one vertebrate species RXR polypeptide fragment andone invertebrate species RXR polypeptide fragment.

In a more preferred embodiment, the chimeric RXR ligand binding domaincomprises at least one vertebrate species RXR polypeptide fragment andone non-Dipteran/non-Lepidopteran invertebrate species RXR homologpolypeptide fragment.

In a specific embodiment, the gene whose expression is to be modulatedis a homologous gene with respect to the host cell. In another specificembodiment, the gene whose expression is to be modulated is aheterologous gene with respect to the host cell.

The ligands for use in the present invention as described below, whencombined with the ligand binding domain of the nuclear receptor(s),which in turn are bound to the response element linked to a gene,provide the means for external temporal regulation of expression of thegene. The binding mechanism or the order in which the various componentsof this invention bind to each other, that is, for example, ligand toligand binding domain, DNA-binding domain to response element,transactivation domain to promoter, etc., is not critical.

In a specific example, binding of the ligand to the ligand bindingdomain of a Group H nuclear receptor and its nuclear receptor ligandbinding domain partner enables expression or suppression of the gene.This mechanism does not exclude the potential for ligand binding to theGroup H nuclear receptor (GHNR) or its partner, and the resultingformation of active homodimer complexes (e.g. GHNR+GHNR orpartner+partner). Preferably, one or more of the receptor domains isvaried producing a hybrid gene switch. Typically, one or more of thethree domains, DBD, LBD, and transactivation domain, may be chosen froma source different than the source of the other domains so that thehybrid genes and the resulting hybrid proteins are optimized in thechosen host cell or organism for transactivating activity, complementarybinding of the ligand, and recognition of a specific response element.In addition, the response element itself can be modified or substitutedwith response elements for other DNA binding protein domains such as theGAL-4 protein from yeast (see Sadowski, et al. (1988) Nature, 335:563-564) or LexA protein from Escherichia coli (see Brent and Ptashne(1985), Cell, 43: 729-736), or synthetic response elements specific fortargeted interactions with proteins designed, modified, and selected forsuch specific interactions (see, for example, Kim, et al. (1997), Proc.Natl. Acad. Sci., USA, 94: 3616-3620) to accommodate hybrid receptors.Another advantage of two-hybrid systems is that they allow choice of apromoter used to drive the gene expression according to a desired endresult. Such double control can be particularly important in areas ofgene therapy, especially when cytotoxic proteins are produced, becauseboth the timing of expression as well as the cells wherein expressionoccurs can be controlled. When genes, operably linked to a suitablepromoter, are introduced into the cells of the subject, expression ofthe exogenous genes is controlled by the presence of the system of thisinvention. Promoters may be constitutively or inducibly regulated or maybe tissue-specific (that is, expressed only in a particular type ofcells) or specific to certain developmental stages of the organism.

The ecdysone receptor is a member of the nuclear receptor superfamilyand classified into subfamily 1, group H (referred to herein as “Group Hnuclear receptors”). The members of each group share 40-60% amino acididentity in the E (ligand binding) domain (Laudet et al., A UnifiedNomenclature System for the Nuclear Receptor Subfamily, 1999; Cell 97:161-163). In addition to the ecdysone receptor, other members of thisnuclear receptor subfamily 1, group H include: ubiquitous receptor (UR),orphan receptor 1 (OR-1), steroid hormone nuclear receptor 1 (NER-1),retinoid X receptor interacting protein-15 (RIP-15), liver X receptor β(LXRβ), steroid hormone receptor like protein (RLD-1), liver X receptor(LXR), liver X receptor α (LXRα), farnesoid X receptor (FXR), receptorinteracting protein 14 (RIP-14), and farnesol receptor (HRR-1

In particular, described herein are novel ligands useful in a geneexpression modulation system comprising a Group H nuclear receptorligand binding domain comprising a substitution mutation. This geneexpression system may be a “single switch”-based gene expression systemin which the transactivation domain, DNA-binding domain and ligandbinding domain are on one encoded polypeptide. Alternatively, the geneexpression modulation system may be a “dual switch”- or“two-hybrid”-based gene expression modulation system in which thetransactivation domain and DNA-binding domain are located on twodifferent encoded polypeptides.

An ecdysone receptor-based gene expression modulation system of thepresent invention may be either heterodimeric or homodimeric. Afunctional EcR complex generally refers to a heterodimeric proteincomplex consisting of two members of the steroid receptor family, anecdysone receptor protein obtained from various insects, and anultraspiracle (USP) protein or the vertebrate homolog of USP, retinoid Xreceptor protein (see Yao, et al. (1993) Nature 366, 476-479; Yao, etal., (1992) Cell 71, 63-72). However, the complex may also be ahomodimer as detailed below. The functional ecdysteroid receptor complexmay also include additional protein(s) such as immunophilins. Additionalmembers of the steroid receptor family of proteins, known astranscriptional factors (such as DHR38 or betaFTZ-1), may also be liganddependent or independent partners for EcR, USP, and/or RXR.Additionally, other cofactors may be required such as proteins generallyknown as coactivators (also termed adapters or mediators). Theseproteins do not bind sequence-specifically to DNA and are not involvedin basal transcription. They may exert their effect on transcriptionactivation through various mechanisms, including stimulation ofDNA-binding of activators, by affecting chromatin structure, or bymediating activator-initiation complex interactions. Examples of suchcoactivators include RIP140, TIF1, RAP46/Bag-1, ARA70, SRC-1/NCoA-1,TIF2/GRIP/NCoA-2, ACTR/AIB 1/RAC3/pCIP as well as the promiscuouscoactivator C response element B binding protein, CBP/p300 (for reviewsee Glass et al., Curr. Opin. Cell Biol. 9:222-232, 1997). Also, proteincofactors generally known as corepressors (also known as repressors,silencers, or silencing mediators) may be required to effectivelyinhibit transcriptional activation in the absence of ligand. Thesecorepressors may interact with the unliganded ecdysone receptor tosilence the activity at the response element. Current evidence suggeststhat the binding of ligand changes the conformation of the receptor,which results in release of the corepressor and recruitment of the abovedescribed coactivators, thereby abolishing their silencing activity.Examples of corepressors include N—CoR and SMRT (for review, see Horwitzet al. Mol. Endocrinol. 10: 1167-1177, 1996). These cofactors may eitherbe endogenous within the cell or organism, or may be added exogenouslyas transgenes to be expressed in either a regulated or unregulatedfashion. Homodimer complexes of the ecdysone receptor protein, USP, orRXR may also be functional under some circumstances.

The ecdysone receptor complex typically includes proteins that aremembers of the nuclear receptor superfamily wherein all members aregenerally characterized by the presence of an amino-terminaltransactivation domain, a DNA binding domain (“DBD”), and a ligandbinding domain (“LBD”) separated from the DBD by a hinge region. As usedherein, the term “DNA binding domain” comprises a minimal polypeptidesequence of a DNA binding protein, up to the entire length of a DNAbinding protein, so long as the DNA binding domain functions toassociate with a particular response element. Members of the nuclearreceptor superfamily are also characterized by the presence of four orfive domains: A/B, C, D, E, and in some members F (see U.S. Pat. No.4,981,784 and Evans, Science 240:889-895 (1988)). The “A/B” domaincorresponds to the transactivation domain, “C” corresponds to the DNAbinding domain, “D” corresponds to the hinge region, and “E” correspondsto the ligand binding domain. Some members of the family may also haveanother transactivation domain on the carboxy-terminal side of the LBDcorresponding to “F”.

The DBD is characterized by the presence of two cysteine zinc fingersbetween which are two amino acid motifs, the P-box and the D-box, whichconfer specificity for ecdysone response elements. These domains may beeither native, modified, or chimeras of different domains ofheterologous receptor proteins. The EcR receptor, like a subset of thesteroid receptor family, also possesses less well-defined regionsresponsible for heterodimerization properties. Because the domains ofnuclear receptors are modular in nature, the LBD, DBD, andtransactivation domains may be interchanged.

Gene switch systems are known that incorporate components from theecdysone receptor complex. However, in these known systems, whenever EcRis used it is associated with native or modified DNA binding domains andtransactivation domains on the same molecule. USP or RXR are typicallyused as silent partners. It has previously been shown that when DNAbinding domains and transactivation domains are on the same molecule thebackground activity in the absence of ligand is high and that suchactivity is dramatically reduced when DNA binding domains andtransactivation domains are on different molecules, that is, on each oftwo partners of a heterodimeric or homodimeric complex (seePCT/US01/09050).

Method of Modulating Gene Expression of the Invention

The present invention also relates to methods of modulating geneexpression in a host cell using a gene expression modulation systemaccording to the invention. Specifically, the present invention providesa method of modulating the expression of a gene in a host cellcomprising the steps of: a) introducing into the host cell a geneexpression modulation system according to the invention; and b)introducing into the host cell a ligand; wherein the gene to bemodulated is a component of a gene expression cassette comprising: i) aresponse element comprising a domain recognized by the DNA bindingdomain of the gene expression system; ii) a promoter that is activatedby the transactivation domain of the gene expression system; and iii) agene whose expression is to be modulated, whereby upon introduction ofthe ligand into the host cell, expression of the gene is modulated.

The invention also provides a method of modulating the expression of agene in a host cell comprising the steps of: a) introducing into thehost cell a gene expression modulation system according to theinvention; b) introducing into the host cell a gene expression cassetteaccording to the invention, wherein the gene expression cassettecomprises i) a response element comprising a domain recognized by theDNA binding domain from the gene expression system; ii) a promoter thatis activated by the transactivation domain of the gene expressionsystem; and iii) a gene whose expression is to be modulated; and c)introducing into the host cell a ligand; whereby upon introduction ofthe ligand into the host cell, expression of the gene is modulated.

The present invention also provides a method of modulating theexpression of a gene in a host cell comprising a gene expressioncassette comprising a response element comprising a domain to which theDNA binding domain from the first hybrid polypeptide of the geneexpression modulation system binds; a promoter that is activated by thetransactivation domain of the second hybrid polypeptide of the geneexpression modulation system; and a gene whose expression is to bemodulated; wherein the method comprises the steps of: a) introducinginto the host cell a gene expression modulation system according to theinvention; and b) introducing into the host cell a ligand; whereby uponintroduction of the ligand into the host, expression of the gene ismodulated.

Genes of interest for expression in a host cell using methods disclosedherein may be endogenous genes or heterologous genes. Nucleic acid oramino acid sequence information for a desired gene or protein can belocated in one of many public access databases, for example, GENBANK,EMBL, Swiss-Prot, and PIR, or in many biology related journalpublications. Thus, those skilled in the art have access to nucleic acidsequence information for virtually all known genes. Such information canthen be used to construct the desired constructs for the insertion ofthe gene of interest within the gene expression cassettes used in themethods described herein.

Examples of genes of interest for expression in a host cell usingmethods set forth herein include, but are not limited to: antigensproduced in plants as vaccines, enzymes like alpha-amylase, phytase,glucanes, and xylanse, genes for resistance against insects, nematodes,fungi, bacteria, viruses, and abiotic stresses, nutraceuticals,pharmaceuticals, vitamins, genes for modifying amino acid content,herbicide resistance, cold, drought, and heat tolerance, industrialproducts, oils, protein, carbohydrates, antioxidants, male sterileplants, flowers, fuels, other output traits, genes encodingtherapeutically desirable polypeptides or products that may be used totreat a condition, a disease, a disorder, a dysfunction, a geneticdefect, such as monoclonal antibodies, enzymes, proteases, cytokines,interferons, insulin, erthropoietin, clotting factors, other bloodfactors or components, viral vectors for gene therapy, virus forvaccines, targets for drug discovery, functional genomics, andproteomics analyses and applications, and the like.

Measuring Gene Expression/Transcription

One useful measurement of the methods of the invention is that of thetranscriptional state of the cell including the identities andabundances of RNA, preferably mRNA species. Such measurements areconveniently conducted by measuring cDNA abundances by any of severalexisting gene expression technologies.

Nucleic acid array technology is a useful technique for determiningdifferential mRNA expression. Such technology includes, for example,oligonucleotide chips and DNA microarrays. These techniques rely on DNAfragments or oligonucleotides which correspond to different genes orcDNAs which are immobilized on a solid support and hybridized to probesprepared from total mRNA pools extracted from cells, tissues, or wholeorganisms and converted to cDNA. Oligonucleotide chips are arrays ofoligonucleotides synthesized on a substrate using photolithographictechniques. Chips have been produced which can analyze for up to 1700genes. DNA microarrays are arrays of DNA samples, typically PCRproducts, that are robotically printed onto a microscope slide. Eachgene is analyzed by a full or partial-length target DNA sequence.Microarrays with up to 10,000 genes are now routinely preparedcommercially. The primary difference between these two techniques isthat oligonucleotide chips typically utilize 25-mer oligonucleotideswhich allow fractionation of short DNA molecules whereas the larger DNAtargets of microarrays, approximately 1000 base pairs, may provide moresensitivity in fractionating complex DNA mixtures.

Another useful measurement of the methods of the invention is that ofdetermining the translation state of the cell by measuring theabundances of the constituent protein species present in the cell usingprocesses well known in the art.

Where identification of genes associated with various physiologicalfunctions is desired, an assay may be employed in which changes in suchfunctions as cell growth, apoptosis, senescence, differentiation,adhesion, binding to a specific molecules, binding to another cell,cellular organization, organogenesis, intracellular transport, transportfacilitation, energy conversion, metabolism, myogenesis, neurogenesis,and/or hematopoiesis is measured.

In addition, selectable marker or reporter gene expression may be usedto measure gene expression modulation using the present invention.

Other methods to detect the products of gene expression are well knownin the art and include Southern blots (DNA detection), dot or slot blots(DNA, RNA), northern blots (RNA), RT-PCR(RNA), western blots(polypeptide detection), and ELISA (polypeptide) analyses. Although lesspreferred, labeled proteins can be used to detect a particular nucleicacid sequence to which it hybidizes.

In some cases it is necessary to amplify the amount of a nucleic acidsequence. This may be carried out using one or more of a number ofsuitable methods including, for example, polymerase chain reaction(“PCR”), ligase chain reaction (“LCR”), strand displacementamplification (“SDA”), transcription-based amplification, and the like.PCR is carried out in accordance with known techniques in which, forexample, a nucleic acid sample is treated in the presence of a heatstable DNA polymerase, under hybridizing conditions, with one pair ofoligonucleotide primers, with one primer hybridizing to one strand(template) of the specific sequence to be detected. The primers aresufficiently complementary to each template strand of the specificsequence to hybridize therewith. An extension product of each primer issynthesized and is complementary to the nucleic acid template strand towhich it hybridized. The extension product synthesized from each primercan also serve as a template for further synthesis of extension productsusing the same primers. Following a sufficient number of rounds ofsynthesis of extension products, the sample may be analyzed as describedabove to assess whether the sequence or sequences to be detected arepresent.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention.

EXAMPLES General Methods

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, N.Y. (1989) (Maniatis) andby T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with GeneFusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984)and by Ausubel, F. M. et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989). All reagents, restriction enzymes andmaterials used for the growth and maintenance of host cells wereobtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories(Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma ChemicalCompany (St. Louis, Mo.) unless otherwise specified.

Manipulations of genetic sequences may be accomplished using the suiteof programs available from the Genetics Computer Group Inc. (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.).Where the GCG program “Pileup” is used the gap creation default value of12, and the gap extension default value of 4 may be used. Where the CGC“Gap” or “Bestfit” program is used the default gap creation penalty of50 and the default gap extension penalty of 3 may be used. In any casewhere GCG program parameters are not prompted for, in these or any otherGCG program, default values may be used.

The meaning of abbreviations is as follows: “h” means hour(s), “min”means minute(s), “sec” means second(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mol” means moles,“mmol” means millimoles, “μg” means microgram(s), “mg” meansmilligram(s), “A” means adenine or adenosine, “T” means thymine orthymidine, “G” means guanine or guanosine, “C” means cytidine orcytosine, “x g” means times gravity, “nt” means nucleotide(s), “aa”means amino acid(s), “bp” means base pair(s), “kb” means kilobase(s),“k” means kilo, “p” means micro, “° C.” means degrees Celsius, “C” inthe context of a chemical equation means Celsius, “THF” meanstetrahydrofuran, “DME” means dimethoxyethane, “DMF” meansdimethylformamide, “NMR” means nuclear magnetic resonance, “psi” refersto pounds per square inch, and “TLC” means thin layer chromatography.

Example 1 Preparation of Compounds

The compounds of the present invention may be made according to thefollowing synthesis routes.

1.1 Preparation of 3,5-Dimethyl-benzoic acidN-tert-butyl-N′-(4-ethyl-2-fluoro-benzoyl)-hydrazide (RG-101523)

To a 3-neck, 2 L round bottom flask was added 173.71 g (1.0 mol, 97%) of2-amino-2-methyl-1-propanol in 300 mL of dry methylene chloride. Theflask was equipped with a magnetic stir bar and thermometer and wasplaced into a dry ice/acetone bath and cooled to 0° C. From a separatoryfunnel, a solution of 4-ethylbenzoyl chloride (168.5 g, 1.0 mol),dissolved in about 300 mL of methylene chloride was slowly added, whilemaintaining the reaction temperature below 5° C. The mixture was allowedto stir at room temperature overnight. Solid propanol amine-HCl wasfiltered off and the filter cake was washed with methylene chloride. Thecombined methylene chloride extracts were concentrated partially on arotary evaporator and used directly in the next step. The intermediateamide solution generated in the first step was cooled in an ice bath andDMF (0.5 mL) was added. 125 g (1.04 mol) of SOCl₂ in 50 mL of methylenechloride from a separatory funnel was added drop-wise at a controlledrate, keeping the reaction temperature at 0-5° C. The reaction wasstirred at room temperature for an additional 2-3 hours. The reactionmixture was cooled in an ice bath and 25% NaOH was added to make theaqueous layer basic (pH=11-12). The mixture was transferred to a largeseparatory funnel, the methylene chloride layer was separated, and theaqueous layer was extracted twice with chloroform. The combined organicphases were dried and evaporated to yield 201 g of2-(4-ethyl-phenyl)-4,4-dimethyl-4,5-dihydro-oxazole as a yellow viscousoil. ¹H NMR (CDCl₃, 300 MHz), .delta. (ppm): 7.8 (d, 2H), 7.2 (d, 2H),4.088 (s, 2H), 2.68 (q, 2H), 1.375 (s, 6H), 1.24 (t, 3H).

The 4-ethylphenyloxazoline (2.03 g, 10 mmol) was dried in a vacuum ovenat 60° C. for 2-3 hours, dissolved in 50 mL of dry THF, and charged to a300 mL round bottom flask equipped with a thermometer, nitrogen inlet,and magnetic stir bar. The mixture was cooled under nitrogen to −70° C.in a dry ice/acetone bath. Butyl lithium in hexane (7.5 mL, 0.012 mmol)was added in two portions and warmed to −25° C. over 2 hours. Themixture was cooled again to 65° C. and N-fluorobenzenesulfonimide (3.79g, 0.012 mmol) was added in three portions. The mixture was allowed towarm to room temperature and was stirred overnight. The reaction mixturewas quenched with 100 mL of saturated NH₄Cl, and transferred to aseparatory funnel with ethyl ether washes. 25% NaOH was added slowly andmixed until an aqueous phase with pH=10 was achieved. The aqueous phasewas extracted with ether and the ether was washed with a small volume ofwater. The ether extracts were dried over MgSO₄ and evaporated to give2.51 g of 2-(2-fluoro-4-ethyl-phenyl)-4,4-dimethyl-4,5-dihydro-oxazoleas a brown oil. In a second experiment, a rate of 70% fluorination wasachieved (highest) with a reactant ratio of 1:1.5:1.5 oxazoline: BuLi:N-fluorobenzenesulfonimide. ¹H NMR (300 MHz, CDCl₃) .delta. (ppm): 7.0(d, 2H), 7.78 (t, 1H), 4.1 (s, 2H), 2.7 (q, 2H), 1.39 (s, 6H), 1.2 (t,3H).

DMSO (4 mL) and CH₃I (2 mL) were added to 2.31 g of oxazoline in a roundbottom flask, and the mixture was stirred overnight at room temperature.Methyl iodide was removed in vacuo on a rotary evaporator. Aqueous KOH(4.4 g in 35 mL of water) was added and the mixture was refluxed for 8hours. The reaction mixture and water washes were transferred to aseparatory funnel; the neutral components were removed with a chloroformextraction. The aqueous mixture was acidified with 6N HCl to pH 1-2, andextracted with ether. Ether extracts were dried over MgSO₄ andevaporated to yield 1.2 g of a white solid, comprised of both2-fluoro-4-ethylbenzoic acid and 4-ethylbenzoic acid. The productmixture was dissolved in KOH and the pH was adjusted with 2N HCl topH=7. With vigorous stirring and careful monitoring with a pH meter, themixture was acidified to pH=5 with 0.1N HCl. 4-Ethylbenzoic acidprecipitated first, which was filtered through Whatman #541 paper,acidification continued with 0.1N HCl to pH=4.9, and at 0.1 unitincrements until pH=4.3, each time filtering the solids through Whatman#541 paper. Finally, the mixture was acidified to pH=2.5 and filtered.Precipitates of decreasing pH contain increasing rations of2-fluoro-4-ethylbenzoic acid, the last two fractions contain 98-100%desired product. Extraction of the remaining aqueous phase with etherrecovers more 2-fluoro-4-ethylbenzoic acid. The product was air-dried asdrying in a vacuum oven results in substantial product losses due tovolatility. ¹H NMR (300 MHz, CDCl₃) δ=7.95 (t, 1H), 7.1 (d, 1H), 7.0 (d,1H), 2.71 (q, 2H), 1.27 (t, 3H). 4-Ethylbenzoic acid: ¹H NMR (CDCl₃) δ(ppm): 8.1 (d, 2H), 7.3 (t, 2H), 2.71 (q, 2H), 1.27 (t, 3H).

1.31 g (1.3 mmol) of thionyl chloride, 1 drop of DMF and 1.0 g (5.95mmol) of acid were added to 30 mL of toluene with stirring. The mixturewas refluxed for 4 hours. After this period, the toluene and unreactedthionyl chloride were removed by distillation. The resulting2-fluoro-4-ethylbenzoyl chloride was used without further purification.

0.150 g of 3,5-dimethyl-benzoic acid N-tert-butyl-hydrazide (1 eq, 0.68mmol) and 0.110 mL (1.2 eq, 0.77 mmol) of 2-fluoro-4-ethyl benzoylchloride were weighed into a 1 oz. vial. A small stirbar was addedfollowed by 2 mL of methylene chloride. The mixture was stirred untilthe hydrazone dissolved. The stirring was stopped and 2 mL of a 1 Mpotassium carbonate (K₂CO₃) solution was added. The mixture was allowedto stir overnight. At the end of this period, 1 mL of water and 1 mL ofmethylene chloride were added. The aqueous phase was removed and theorganic phase was washed twice with 1 M potassium carbonate solution.The organic phase was removed and dried over magnesium sulfate. Theorganic phase was filtered thru a pad of basic alumina and the solventremoved. The product, 3,5-dimethylbenzoic acidN-tert-butyl-N′-(2-fluoro-4-ethylbenzoyl)-hydrazide, was purified bytrituration with 1:1 ether:hexane. ¹H NMR (300 MHz, CDCl₃) δ (ppm): 7.7(m, 2H), 7.6 (t, 1H), 7.0 (m, 3H), 2.6 (q, 2H), 2.3 (s, 3H), 2.1 (s,6H), 1.5 (s, 9H), 1.1 (t, 3H).

1.2 Preparation of 5-chloro-4H-benzo[1,3]dioxine-6-carboxylic acid

5-chloro and 7-chloro isomers were separated by silica gel cartridgechromatography. The mixture was dissolved in CHCl₃/CH₃OH and added tothe top of a large cartridge. The 5-chloro isomer eluted with 2:3ether:hexane and the 7-chloroisomer began to elute with 3:2 ether:hexaneand completed elution with neat ether. ¹H NMR (DMSO-d₆, 300 MHz) δ(ppm): 7.75 (d, 1H), 6.95 (d, 1H), 5.3 (s, 2H), 4.9 (s, 2H).

1.3 Preparation of 2-fluoro-4-hydroxy-benzoic acid

To a stirred solution of 2-fluoro-4-hydroxybenzonitrile (20.00 g, 145.9mmol) in 160 mL of water, was added 50% aqueous sodium hydroxide (40.00g, 500.0 mmol). The mixture was heated to reflux for 4 hours, cooled toroom temperature, poured into iced concentrated hydrochloric acid, andextracted with ether. The product was extracted into saturated aqueoussodium bicarbonate and the ether layer discarded. This aqueous extractwas acidified with concentrated hydrochloric acid and extracted withether. The organic extract was dried over magnesium sulfate, filtered,and evaporated to give a white solid (22.90 g) of2-fluoro-4-hydroxybenzoic acid in 100% yield. ¹H NMR (300 MHz, CD₃COCD₃)δ (ppm): 9.80 (b, 1H), 7.87 (t, 1H), 6.77 (dd, 1H), 6.66 (dd, 1H).¹⁹F-NMR (300 MHz, CD₃COCD₃) δ (ppm): −108.13 (s, decoupled).2-Fluoro-4-hydroxybenzonitrile: ¹H-NMR (300 MHz, CD₃COCD₃) δ (ppm): 7.61(t, 1H), 6.81 (m, 2H), 5.80 (b, 1H). ¹⁹F-NMR (300 MHz, CD₃COCD₃) δ(ppm): −108.82 (s, decoupled)

1.4 Preparation of 5-fluoro-4H-benzo[1,3]dioxine-6-carboxylic acid,methyl ester and 6-fluoro-4H-benzo{1,3}dioxine-7-carboxylic acid, methylester

1.6 g methyl 2-fluoro-4-hydroxy-benzoate (7.54 mmol), 0.157 g ofp-toluenesulfonic acid (0.9 mmol), 50 mL of toluene and 1.2 g ofparaformaldehyde (40 mmol) were combined and refluxed for 3 hours afterwhich time TLC (1:1 ethyl acetate: CH₂Cl₂) showed the absence of thestarting material. Occasionally it became necessary to cool the reactionand scrape off unreacted paraformaldehyde from the walls of the reactionflask. The reaction flask was vented to the hood exhaust. After themixture was filtered to remove the solid paraformaldehyde, the solid waswashed twice with 100 mL of toluene. The toluene washes were combinedwith the filtered liquid. The organic liquid was washed three times with75 mL of 5% aqueous NaOH. 50 mL of methanol was added to the organicphase and the solvent was removed on the rotary evaporator to yield athick syrup which gradually formed a somewhat tacky white solid. Protonand ¹⁹F NMR showed the presence of two isomers in a ratio ofapproximately 7:3. These isomers could be separated by carefulchromatography on silica gel using a hexane to 85% hexane-15% ethergradient. The desired 3,4-methylenedioxy-2-fluoro benzoic acid, methylester eluted first as a white solid. ¹H NMR (CDCl₃, 300 MHz) δ (ppm):3.85 (s, 3H), 4.85 (s, 2H), 5.20 (s, 2H), 6.60 (d, 1H), 7.80 (t, 1H).¹⁹F NMR (ppm, CDCl₃)-115 (s); Rf=0.4 (1:1 CH₂Cl₂: EtOAc).

The 4,5-methylenedioxy isomer eluted shortly thereafter, also as a whitesolid. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 3.90 (s, 3H), 4.85 (s, 2H), 5.25(s, 2H), 6.65 (d, 1H), 7.60 (d, 1H). ¹⁹F NMR (ppm, CDCl₃)-108 (s).);Rf=0.32 (1:1 CH₂Cl₂: EtOAc).

1.5 Preparation of 5-fluoro-4H-benzo{1.3}dioxin-6-carboxylic acid and6-fluoro-4H-benzo{1,3}dioxin-7-carboxylic acid

5-Fluoro-4H-benzo[1,3]dioxine-6-carboxylic acid methyl ester (2.02 g),water (1 mL), methanol (20 mL) and sodium hydroxide (1 mL of a 50% NaOHsolution) were added to a flask equipped with a condenser and magneticstirbar. The stirring was started and the mixture was refluxed for 2hours. At this point, the TLC (1:1: CH₂Cl₂/ethyl acetate) showed nostarting ester was present. The solvent was removed, leaving a whitesolid. The solid was taken up in water and the aqueous layer was washedthree times with 50 mL of ether. The aqueous layer was then acidifiedwith dilute hydrochloric acid causing the formation of a whiteprecipitate. This white solid was collected on a sintered glass filterfunnel and washed well with de-ionized water. The solid was dried undervacuum at 60° C. overnight and used in the following reaction withoutfurther purification.

1.6 Preparation of 5-fluoro-4H-benzol{1,3}dioxine-6-carboxylic acidN′-tert-butyl-hydrazide and 5-fluoro-4H-[1,3]dioxine-6-carboxlyic acidN-tert-butyl-N′-(5-fluoro-4H-benzo[1,3]dioxine-6-carbonyl)-hydrazide

1.6 g of 5-fluoro-4H-benzo{1.3}dioxin-6-carboxylic acid (8.1 mmol), 30mL of toluene and 1 drop of DMF were combined in a 100 mL flask equippedwith magnetic stirbar, scrubber and condenser. 0.59 mL of thionylchloride (0.96 g, 9.7 mmol) was added and the mixture was heated toreflux and held at reflux for 4 hours. After this period, the mixturewas cooled slightly and the condenser was replaced with a distillationhead. The excess thionyl chloride was distilled off. The mixture wascooled to 20° C. and the toluene was removed using a rotary evaporator.NOTE: It is advisable to use care during the toluene removal. The5-fluoro-4H-benzo[1,3]dioxine-6-carbonyl chloride starts to distillunder vacuum if the temperature exceeds 27° C. 50% NaOH (0.648 g, 8.1mmol) of was dissolved in 3 mL of water and added to a reaction flaskcontaining a magnetic stirbar and rubber septum for reagent addition.1.00 (8.1 mmol) g of tert-butyl hydrazine hydrochloride was added. Themixture was stirred for 5 min at room temperature and then cooled to −5°C. 5-Fluoro-4H-benzo[1,3]dioxine-6-carbonyl chloride (8.1 mmol) wasdissolved in 25 mL of dichloromethane and was added simultaneously witha second portion of 0.648 g (8.1 mmol) of 50% NaOH in 3 mL of water. Thereaction temperature was kept below −2° C. during the addition. Themixture was stirred at −5° C. to −2° C. for 30 min. After this time, themixture was allowed to warm to room temperature and stirred for 30 min.50 mL of dichloromethane and 50 mL of water were added to the reactionmixture. The layers were separated and the organic layer was washedthree times with 50 mL of water. The organic layer was then dried overMgSO₄ and filtered. Removal of the solvent yielded 2.4 g of a yellowsyrup, which appeared to be approximately 85% of the desired product byNMR analysis. The pure product,5-fluoro-4H-benzo[1,3]dioxine-6-carboxylic acid N′-tert-butyl-hydrazide,was isolated as a pale, yellow solid by careful column chromatography onsilica gel using a dichloromethane to 4:1 dichloromethane/ethyl acetategradient. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 0.85 (s, 9H), 4.58, (s, 2H),4.90 (s, 2H), 6.40 (d, 1H), 7.50 (t, 1H) and 7.70 (br s, 1H). ¹⁹F NMR(CDCl₃), δ ppm: (s, −190, s). Rf=0.35 (1:1 CH₂Cl₂:ethyl acetate).

1.7 Preparation of 2-Bromomethyl-3-methoxy-benzoic acid methyl ester

Into a 2 L 3-neck round bottom flask was added 75 g (0.42 mol) of2-methyl-3-methoxy methyl benzoate, 500 mL of CCl₄, 80.1 g (0.45 mol) ofNBS, and 1 g of AIBN. The mixture was stirred and refluxed gently for 2hours. The reaction mixture was cooled and ca. 600 mL of CH₂Cl₂ and 500mL of water were added. The mixture was stirred to dissolve floatingsolids, transferred to a 2 L separatory funnel, and then shaken. Theorganic layer was separated and the water extracted with CH₂Cl₂. Theaqueous fractions were discarded and the organic phase extracted with400-500 mL of water to remove the NBS (note: solubility of succinimideis 1 g/3 g water, solubility of NBS is 1.47 g/100 mL water). The waterextractions were repeated, the organic phase dried with MgSO₄ andcharcoal, and the solvent evaporated in 2 portions, to yieldmethyl-3-methoxy-2-bromomethylbenzoate. TLC: Rf=0.58, single spot (1:1ethyl acetate:hexane). ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.5 (d, 1H), 7.3(t, 1H), 7.05 (d, 1H), 5.06 (s, 2H), 3.94 (s, 3H), 3.93 (s, 3H).

1.8 Preparation of 4-methoxy-3H-isobenzofuran-1-one

In a 500 mL round bottom flask was added 15.15 g (0.0585 mol) of2-bromomethyl, 3-methoxy methyl benzoate, 29.3 g (0.293 mol) of CaCO₃,150 mL of dioxane and 150 mL of water. The flask was placed into an oilbath and the mixture heated with stirring at 85° C. for 3.5 to 4 hours.The CaCO₃ was filtered off and washed with ethyl acetate and water. Tothe filtrate was added ethyl acetate (200 mL) and water (50 mL) and themixture then shaken in separatory funnel. The water phase was extractedtwice with ethyl acetate (50 mL). The ethyl acetate extracts werecombined, extracted once with water, dried over MgSO₄, and evaporated.This yielded 9.2 g of white crystals of 7-methoxybenzolactone (95%yield). ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.5 (m, 2H), 7.1 (m, 1H), 5.26(s, 2H), 3.93 (s, 3H). TLC: Rf=0.46 (1:1 EtOAC:hexane).

1.9 Preparation of 2-cyanomethyl-3-methoxybenzoic acid

Into a 500 mL 3-neck round bottom flask was added 10 g (61.75 mmol) of2-bromomethyl, 3-methoxy methyl benzoate, 4.0 g (81.6 mmol) of NaCN,0.30 g (2 mmol) of NaI, 100 mL of CH₃CN, and 50 mL of DMF. The reactionmixture was heated and refluxed for 10 hours. The precipitate (NaBr) wasfiltered off, and the solution was concentrated on an evaporator. 300 mLof water and 200 mL of ether were added and then shaken in a separatoryfunnel. The water was extracted twice with 100 mL of ether. The etherfractions were dried over MgSO₄, and concentrated to yield methyl3-methoxy-2-cyanomethylbenzoate (95-100% yield). This ester (0.053 mmol,10.51 g) was stirred vigorously in 100 mL of CH₃OH. Ba(OH)₂H₂O (0.079mmol, 14.97 g) was added and the mixture stirred at room temperatureovernight. The CH₃OH was removed on a rotary evaporator. 150 mL ofwater, 200 mL of CH₂Cl₂, and 50 mL of 6N HCl were added, and thenstirred in a flask to dissolve all residues. The mixture was transferredto a separatory funnel, acidified with 6N HCl to pH 1-2. The CH₂Cl₂phase was separated and the aqueous phase extracted twice with 50 mL ofCH₂Cl₂. The CH₂Cl₂ extracts were combined, dried over MgSO₄ andcharcoal, filtered, and evaporated to yield 8.8 g of a white solid,2-cyanomethyl-3-methoxybenzoic acid, (87%).

Methyl 3-methoxy-2-cyanomethylbenzoate: ¹H NMR (CDCl₃, 300 MHz) δ (ppm):7.6 (d, 1H), 7.4 (t, 1H), 7.1 (d, 1H), 4.18 (s, 2H), 3.94 (s, 3H), 3.926(s, 3H). TLC (1:1 ethyl acetate:hexane) 0.55.

2-Cyanomethyl-3-methoxybenzoic acid: ¹H NMR (300 Mhz, CDCl₃) δ (ppm):7.55 (d, 1H), 7.45 (t, 1H), 7.3 (d, 1H), 4.121 (d, 2H), 3.91, (s, 3H).TLC (1:1 ethyl acetate:hexane), Rf 0.36 streak.

1.10 Preparation of 3-methoxy-2-methylsulfanylmethyl-benzoic acid andpentafluorophenyl 2-(methylthiomethyl 3-methoxybenzoate

Methyl 2-bromomethyl-3-methoxy benzoate was stirred in methanol at roomtemperature with 1.02 eq. of sodium methylmercaptide. After 30 min thereaction was complete based on GC analysis. The mixture was poured intowater and extracted twice with ethyl acetate. The combined organiclayers were stripped under vacuum leaving methyl2-(methylthiomethyl)-3-methoxybenzoate as a pale yellow oil in about 86%yield. GC: DB-5, 30 m, film: 0.25 um, t_(init)=1.00 T=120-280C@20C/min;Rt=6:30 area %=98. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.46 (d, 1H), 7.26(t, 1H), 7.03 (d, 1H), 4.18 (s, 1H), 3.90 (s, 2H), 3.89 (s, 3H), 2.04(s, 3H).

Methyl 2-(methylthiomethyl)-3-methoxy benzoate was heated to reflux with1.5 eq of NaOH in 10% aqueous methanol for 1.5 hr. The solution wasadded drop-wise to excess 10% sulfuric acid. The precipitate wasfiltered and dried in air giving ca. 92% yield of2-(methylthiomethyl)-3-methoxybenzoic acid. TLC (2:1 ethylacetate:hexane) indicated one spot, Rf 0.50.

2-(Methylthiomethyl)-3-methoxybenzoic acid was dissolved in ethylacetate and added to a solution of 1.1 eq. of pentafluorophenol and 1.1eq. of dicyclohexycarbodiimide in ethyl acetate. After 1 hr the mixturewas filtered and the mother liquors were stripped under vacuum. Theyellow oily residue was crystallized from hexane to give the product,pentafluorophenyl 2-(methylthiomethyl 3-methoxybenzoate, in 100% yield.TLC (1:2 ethyl acetate:hexane) indicated one spot, Rf 0.58.

1.11 Preparation of 3-Methoxy-2-methoxymethyl-benzoic acid

To a 100 mL flask containing 10.1 g (0.039 mol) of methyl2-bromomethyl-3-methoxybenzoate in 50 mL of CH₃OH, were added 19 g of a25% wt. solution of NaOMe (4.74 g, 0.087 mol). The reaction was stirredat room temperature for 2 hours and then evaporated on a rotaryevaporator to remove the CH₃OH. About 200 mL of water were added to theresidue and the resulting solution was extracted with CHCl₃. The CHCl₃extract was dried and evaporated to yield 6.27 g of crude methyl3-methoxy-2-methoxymethylbenzoate (77% yield). ¹H NMR (CDCl₃, 300 MHz) δ(ppm): 7-7.4 (multiple, 3H), 4.783 (s, 2H), 3.897 (s, 3H), 3.864 (s,3H), 3.37 (s, 3H).

6.27 g of methyl 3-methoxy-2-methoxymethylbenzoate was stirred with a20% aqueous KOH solution (6.7 g, 0.12 mol in 34 g of solution) at 50° C.in an oil bath for 4-5 hours, and then at room temperature for 16 hours.The reaction mixture was acidified with 3N HCl to pH 2 and extractedwith CH₂Cl₂. The CH₂Cl₂ extract was dried and evaporated to yield 5.35 gof 3-methoxy-2-methoxymethylbenzoic acid (92% yield). ¹H NMR (CDCl₃, 300MHz) δ (ppm): 7.65 (1H, d), 7.40 (t, 1H), 7.10 (d, 1H), 4.83 (s, 2H),3.88 (s, 3H), 3.46 (s, 3H).

1.12 Preparation of N′-t-butyl-3-methoxy-2-methoxymethylphenylhydrazide

To 4.30 g (0.0219 m) of 3-methoxy-2-methoxymethylbenzoic acid in 50 mLof ethyl acetate in a round bottom flask, was added 8.88 g of a 50% wtsolution of pentafluorophenyl phenol, followed by 21.91 mL of DCCsolution (0.0219 m). After stirring for 2 hr at room temperature, TLCshowed a spot for the intended pentafluorophenyl ester product atRf=0.64 (1:1 ethyl acetate:hexanes), while the starting acid wasRf=0.39.

A small volume of ethyl acetate (30 mL) and a teaspoon of anhydrousMgSO₄ was added and then filtered to remove the DCC and DCU. Thefiltrate was evaporated to yield 9.4 g of product. ¹H NMR (CDCl₃, 300MHz) δ (ppm): 3.39 (s, 3H), 3.90 (s, 3H), 4.81 (s, 2H). NMR analysis ofthe starting material indicated the following spectrum: ¹H NMR (CDCl₃,300 MHz) δ (ppm): 4.83 (s, 2H), 3.88 (s, 3H), 3.4 (s, 3H).

The remaining DCC and DCU were removed by column chromatography onsilica gel. The products eluted in the 6 and 8% ethyl acetate/hexanefractions. The yield of 7.08 g, contained some DCC and DCU. 7.08 g (0.02mol) of the pentafluorophenyl ester in 60 mL of CH₂Cl₂ was stirred with3.67 g (0.029 mol) of t-butylhydrazine HCl and 12 g of K₂CO₃ in 60 mL ofwater at room temperature overnight. 60 mL of CH₂Cl₂ and 50 mL of waterwere added and then shaken in separatory funnel. The organic phase wasdried over MgSO₄ and then evaporated to yield 5.6 g of t-butylhydrazide. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.4 (d, 2H). 7.0 (t, 1H),4.627 (s, 2H), 3.874 (s, 3H), 3.465 (s, 3H), 1.184 (s, 9H). TLC: (1:1ethyl acetate:hexanes), Rf=0.16. The product can be further purified bytrituration with hexanes.

1.13 Preparation of 3-methoxy-2-methoxymethyl-benzoic acid

1.0 g (0.0061 mol) of lactone was refluxed with 20 mL of 7.5% NaOH and20 mL of CH₃OH for 7 hours. The methanol was removed on a rotaryevaporator, set up a Dean Stark, refluxed in toluene to azeotropicallyremove water, toluene was removed in vacuo, and the residue was dried ina vacuum oven. ¹H NMR (DMSO, 300 MHz) δ (ppm): 3.74 (s, 3H), 4.47 (s,2H), 6.9 (d, 1H), 7.1 (t, 1H), 7.2 (d, 1H).

The sodium carboxylate was dissolved in 15 mL of DMF and then CH₃I (0.87g, 0.0061 mol) was added and the mixture was stirred at room temperatureovernight. 50 mL of saturated NH₄Cl was added to quench the reaction.250 mL of water was added (solution is basic at this point), andextracted with ether to remove the neutral and basic substances. Theremaining aqueous solution was acidified with 3N HCl to pH=2 and thedesired carboxylic acid extracted with ether. The ether extracts weredried and evaporated to yield 0.55 g of3-methoxy-2-hydroxymethylbenzoate, sodium salt and3-methoxy-2-methoxymethylbenzoic acid. ¹H NMR (CDCl₃, 300 MHz) δ (ppm):3.456 (s, 3H), 3.884 (s, 3H), 4.832 (s, 2H), 7.1 (d, 1H), 7.4 (t, 1H),7.6 (d, 1H).

1.14 Preparation of 2-allyloxymethyl-3-methoxy-benzoic acid

1.0 g (0.005 mol) of sodium 3-methoxy-2-hydroxymethylbenzoate wascombined in a 200 mL flask with 1.68 g (0.01 mol) of allyl iodide and 50mL of dioxane and refluxed for 2 hr. The mixture was stirred at roomtemperature overnight. The reaction mixture was concentrated on anevaporator. Water were added, and aqueous 5% NaOH to pH=10-11, and themixture was extracted with ether. The ether was evaporated to give adiallyl product (0.34 g). ¹H NMR indicated complex allyl signals inaddition to the aromatic protons. The water solution was acidified with3N HCl and extracted twice with 100 mL of ether to yield3-methoxy-2-allyloxybenzoic acid and allyl 3-methoxy-2-allyloxybenzoate(0.34 g). ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 3.879 (s, 3H), 4.11 (d, 2H),5.3 (q, 2H), 5.9-6.0 (m, 1H). TLC: (1:1 ethyl acetate:hexane): diallyl,Rf 0.60, monoallyl, Rf 0.31, streak.

1.15 Preparation of methyl 3-methoxy-2-allyloxymethyl benzoate

Into a 25 mL round bottom flask, was added 0.96 g (0.0165 m) of allylalcohol and 3 mL of DMF. While cooling the flask in an ice bath, 0.80 gof a 60% dispersion of NaH (0.020 m, 0.48 g) was added, with magneticstirring. The reaction mixture was stirred for 45 min at roomtemperature. The flask was placed in the ice bath and 2 g of DMF and3.89 g (0.015m) of methyl 2-bromomethyl-3-methoxy benzoate were added insmall portions. The reaction was allowed to stir at room temperature for4-5 hours. The reaction was transferred to a separatory funnel with 150mL of ethyl ether and 50 mL of water. The reaction mixture was shaken,the ether phase separated and the water phase again extracted with 50 mLof ether. The ether phase was extracted with water (20 mL), dried withMgSO₄ and concentrated to yield 2.7 g of a pale, yellow oil (76% yield).¹H NMR (CDCL₃, 300 MHz) δ (ppm): 7.3-7.0 (m, 3H), 5.9-6.0 (m, 1H),5.1-5.3 (2d, 2H), 4.8 (d, 2H), 4.02 (d, 2H), 3.90 (s, 3H), 3.88 (s, 3H).TLC (1:1 ethyl acetate/hexane), Rf 0.58.

1.16 Preparation of 2-allyloxymethyl-3-methoxybenzoic acid

Into a 200 mL round bottom flask containing 5.40 g (0.0229 m) of3-methoxy-2-allyloxymethyl benzoate, was added 40 mL of methyl-alcohol.With magnetic stirring, 6.50 g (0.034 m) of barium hydroxide monohydratewas added. The reaction was stirred for 4 hours in a 45° C. water bath.The reaction flask was transferred to a rotary evaporator and themethanol was removed under vacuum. H₂O (150 mL) was added to the residuein the flask and the mixture was stirred until most of the residuedissolved. The reaction mixture was transferred with water (50-100 mL)to a large beaker. The mixture was acidified with 6HCl (to pH=1) andtransferred to a separatory funnel. The reaction mixture was extractedthree times with 100 mL of ethyl acetate with salting out. Ethyl acetateextract was dried and evaporated to yield 4.38 (g) of viscous product,2-allyloxymethyl-3-methoxybenzoic acid (98% yield). ¹H NMR (CDCL₃, 300MHz) δ (ppm): 7.55 (d, 1H), 7.40 (t, 1H), 7.1 (d, 1H), 6.0-5.9 (m, 1H),5.4-5.2 (2d, 2H), 4.87 (d, 2H), 4.10 (d, 2H), 3.878 (s, 3H). TLC(1:1ethyl acetate:hexane) Rf 0.38.

1.17 Preparation of pentafluorophenyl 2-allyloxymethyl-3-methoxybenzoate

Into a 200 mL round bottom flask was added 6.6 g (0.0297 mol) of2-allyloxymethyl-3-methoxy benzoic acid and 40 mL of ethyl acetate.24.05 g of a 25% pentafluorophenol (6.01 g, 0.0327 mol) solution inethyl acetate was added while stirring. The reaction flask was placedinto a water bath and while stirring small portions of DCC (6.2 g, 0.030mol) were added. The stirring continued overnight at room temperature.The reaction was filtered through two Whatman #541 filters to remove theDCU precipitate. The ethyl acetate solution was concentrated to yield12.8 g (110% yield), indicating presence of DCC and DCU. This wasconfirmed by TLC (1:1 ethyl acetate:hexane), which indicated a Rf of0.72 plus other less polar compounds (12 stain indicates about 85%purity). ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.7 (d, 1H), 7.45 (t, 1H),7.15 (d, 1H), 6.0-5.9 (m, 1H), 5.3-5.1 (2d, 2H), 4.88-4.83 (d, 2H), 4.06(d, 2H), 3.90 (s, 3H).

1.18 Preparation of 2-allyloxymethyl-3-methoxy-benzoic acidN′-tert-butyl-hydrazide

Into a round bottom flask containing 18.9 g (0.048 m) ofpentafluorophenyl ester in 50 mL of CH₂Cl₂, was added 9.1 g (0.73 m) oft-butylhydrazine hydrochloride, and then 20.16 g (0.146 m) of K₂CO₃ in50 mL of H₂O. The mixture was stirred at room temperature for 24 hours.50 mL of H₂O were added, the CH₂Cl₂ layer was separated, and H₂O phaseextracted twice with 100 mL of CH₂Cl₂. The CH₂Cl₂ fraction was driedwith MgSO₄, and concentrated to yield 9.75 g ofN-2-allyloxymethyl-3-methoxyphenyl-N′-t-butylhydrazide. ¹H NMR (CDCl₃,300 MHz) δ (ppm): 1.151 (s, 9H), 3.87 (s, 3H), 4.12 (d, 2H), 4.68 (s,2H), 5.15-5.35 (q, 2H), 5.19 (m, 1H), 7.0 (t, 1H), 7.4 (d, 2H). TLC:(1:1, ethyl acetate:hexane) Rf=0.25.

1.19 Preparation of 3,5-dimethyl-benzoic acidN′-(2-allyloxymethyl-3-methoxy-benzoyl)-N-tert-butyl-hydrazide(RG-115003)

To a flask containing 2.0 g (0.0068 mol) of2-allyloxymethyl-3-methoxy-benzoic acid N′-tert-butyl-hydrazidedissolved in 15 mL of CH₂Cl₂, was added 1.27 g (0.0075 mol) of3,5-dimethylbenzoyl chloride in 10 mL of CH₂Cl₂ and 2.84 g of K₂CO₃(0.02 mol) in 30 mL of H₂O. The mixture was stirred at room temperaturefor 24 hours. The reaction mixture was diluted and partitioned, and theorganic phase was dried and solvent was removed in vacuo. The productwas purified by silica gel chromatography; eluting in 25% ethylacetate:hexane fractions, to yield 2.40 g of pure 3,5-dimethyl-benzoicacid N′-(2-allyloxymethyl-3-methoxy-benzoyl)-N-tert-butyl-hydrazide. ¹HNMR (CDCl₃, 300 MHz) δ (ppm): 7.2 (t, 1H) 7.1 (s, 1H), 7.05 (d, 2H),6.95 (m, 2H), 5.9 (m, 1H), 5.2-5.3 (q, 2H), 4.5 (d, 2H), 3.9 (m, 2H),3.80 (s, 3H), 2.245 (s, 6H), 1.547 (s, 9H).

1.20 Preparation of 3,5-dimethyl-benzoic acidN-tert-butyl-N′-(2-hydroxymethyl-3-methoxy-benzoyl)-hydrazide(RG-115371)

1.57 g of allyl ether were dissolved in 50 mL of CH₃OH. 600 mg of Pd/Cand 20 drops of 1% HClO₄/H₂O were added and refluxed for 4 hours. CH₂Cl₂(70 mL) and a teaspoon of anhydrous MgSO₄ were added and then filtered.The filtrate was evaporated to dryness to yield 1.62 g of crude benzylicalcohol. The product was purified by column chromatography on silica geland eluted with 50-60% ethyl acetate/hexanes to yield 1.25 g of3,5-dimethyl-benzoic acidN-tert-butyl-N′-(2-hydroxymethyl-3-methoxy-benzoyl)-hydrazide as a whitesolid. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.1 (t, 1H), 7.05 (s, 2H), 6.98(s, 1H), 6.9 (d, 1H), 6.5 (d, 1H), 4.2 (q, 2H), 3.79 (s, 3H), 2.23 (s,6H), 1.57 (s, 9H). TLC (1:1 ethyl acetate:hexane) Rf=0.20.

1.21 Preparation of 3,5-dimethyl-benzoic acidN-tert-butyl-N′-(2-chloromethyl-3-methoxy-benzoyl)-hydrazide (RG-115490)

To a 50 mL round bottom flask, was added 400 mg (0.00315 mol) of oxalylchloride and 5 mL of CH₂Cl₂. The mixture was stirred and then cooled inacetone/dry ice bath to −70° C. 616-620 mg (0.0079 mol) of DMSO in 5 mLof CH₂Cl₂ was slowly added and stirred for 30 min at −70° C. 405 mg(0.00105 mol) of RG-115371 in 4 mL of CH₂Cl₂ was added and stirred for30 min at −70° C. The dry ice bath was removed and the mixture wasallowed to warm to room temperature over 30 min. The mixture was coolagain to −70° C. and then 1.60 g (0.158 mol) of triethylamine was addedand the mixture was allowed to warm to room temperature. 6 mL of waterwas added to quench the reaction. CH₂Cl₂ was added to the flask andtransferred to a separatory funnel with a total of 100 mL of CH₂Cl₂. 50mL of water were added and the aqueous layer was again extracted withCH₂Cl₂. The CH₂Cl₂ extract was extracted with dilute (0.05-0.1 N)HCl/H₂Oto remove the Et₃N and DMSO. The CH₂Cl₂ extract was dried andconcentrated to yield about 0.44 g of product. TLC: Rf=0.47. The productcan be purified by silica gel column chromatography, eluting with 35-40%ethyl acetate in hexanes. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 8.0 (s, 1H),6.9-7.2 (t,s,s,d, 5H), 6.35 (d, 1H), 4.5 (d, 1H), 4.1 (d, 1H), 3.85 (s,3H), 2.28 (s, 6H), 1.59 (s, 9H).

1.22 Preparation of 3,5-dimethyl-benzoic acidN-tert-butyl-N′-(2-iodomethyl-3-methoxy-benzoyl)-hydrazide

To a 50 mL flask containing 400 mg of the RG-115490, was added 10 mL ofCH₃CN, 1 mL of DMF, and 100 mg of NaI. The mixture was refluxed for 4hours. The reaction was poured into 250 mL of ether and 75 mL of waterin a separatory funnel. The mixture was shaken vigorously and the etherlayer extracted with ca. 50 mL of water. The ether extracts were driedover MgSO₄ and charcoal, filtered, and the solvent removed to yield 250mg of a yellow solid. TLC Rf=0.50 (1:1 ethyl acetate:hexane). ¹H NMR(CDCl₃, 300 MHz) δ (ppm): 6.9-7.3 (m, 6H), 6.3 (d, 1H), 4.3 (d, 1H), 4.2(d, 1H), 3.88 (s, 3H), 2.28 (s, 6H), 1.62 (s, 9H).

1.23 Preparation of 3,5-dimethyl-benzoic acidN-tert-butyl-N′-(2-methylaminomethyl-3-methoxy-benzoyl)-hydrazide(RG-115079)

Into a small flask, containing 300 mg of the RG-115490 dissolved in 15mL of dioxane (99.8% anhydrous, Aldrich), was added CH₃NH₂ in dioxane (4eq). The reaction was refluxed for 2 hours. The solvent was removed on arotovap, redissolved in CH₂Cl₂, filtered, and the CH₂Cl₂ solubles wereconcentrated. The methylamine was obtained after column chromatographyby elution with ethyl acetate, then 9:1 ethyl acetate: methanol withabout 0.2% triethylamine, after having run the column with 4:1 ethylacetate:hexane, 0.2% triethylamine. The total yield of the product was183 mg. TLC: Rf 0.23 (1:1 ethyl acetate:hexane+triethylamine). ¹H NMR(CDCl₃, 300 MHz), δ (ppm): 7.3-6.9 (m, 6H), 3.80 (s, 3H), 3.6 (d, 1H),2.8 (d, 1H), 2.4 (s, 3H), 2.28 (s, 6H), 1.59 (s, 9H).

1.24 Preparation of 3,5-dimethyl-benzoic acidN-tert-butyl-N′-(2-dimethylaminomethyl-3-methoxy-benzoyl)-hydrazide(RG-115079)

250 mg (0.0006 mol) of the RG-115490 was added to a 20 mL vial. 3 mL ofTHF and 0.31 mL of a 2 M dimethylamine/THF solution (Aldrich) was thenadded. The mixture was stirred for 4 hr at room temperature. The solventwas removed on a rotovap and the solid was triturated with hexane whilestirring at room temperature. 3,5-Dimethyl-benzoic acidN-tert-butyl-N′-(2-dimethylaminomethyl-3-methoxy-benzoyl)-hydrazide: ¹HNMR (CDCl₃, 300 MHz) δ (ppm): 7.1-6.9 (m, 6H), 3.93 (s, 3H), 2.68 (s,3H), 2.54 (s, 3H), 2.24 (s, 6H), 1.61 (s, 9H).

1.25 Preparation of 3,5-dimethyl-benzoic acidN-tert-butyl-N′-(2-acetoxymethyl-3-methoxy-benzoyl)-hydrazide(RG-115225)

In a 20 mL vial containing 200 mg of RG-115371 in 4 mL of anhydrousCH₂Cl₂, was added 200 mg of Et₃N and 10 mg of CH₃COCl. The mixture wasstirred at room temperature overnight. TLC indicated an incompletereaction. 100 mg of acetyl chloride and some pyridine were added andrefluxed for 1 hour. The reaction mixture was poured into CH₂Cl₂ andextracted with aqueous, dilute K₂CO₃, then dilute aqueous HCl. TheCH₂Cl₂ extract was dried and concentrated, to yield a crude acetate. Thematerial was purified by silica gel column chromatography, eluting with1:1 ethyl acetate:hexane. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.3-6.9 (m,6H), 6.4 (d, 1H), 4.8 (d, 1H), 4.6 (d, 1H), 3.795 (s, 3H), 2.3 (s, 6H),2.03 (s, 3H), 1.58 (s, 9H).

1.26 Preparation of 2-methanesulfinylmethyl-3-methoxy-benzoic acidN′-tert-butyl-N′-(3,5-dimethyl-benzoyl)-hydrazide (RG-115172)

3-Methoxy-2-methylsulfanylmethyl-benzoic acidN′-tert-butyl-N′-(3,5-dimethyl-benzoyl)-hydrazide in CH₂Cl₂ was stirredat room temperature with 1.0 eq of m-chloroperbenzoic acid. The reactionwas complete within 5 min as indicated by TLC. The reaction mixture waswashed with saturated NaHCO₃ and the organic layer was stripped undervacuum. The residue was mixed with 1-2 mL of 1:1 ether:hexane and thesolution was removed with a pipette, leaving the product which was thendried under vacuum.

1.27 Preparation of 2-methanesulfonylmethyl-3-methoxy-benzoic acidN′-tert-butyl-N′-(3,5-dimethyl-benzoyl)-hydrazide (RG-115408)

RG-115172 was dissolved in ethylene dichloride. 1.2 eq. ofm-chloroperbenzoic acid was added and the mixture was heated to reflux.The reaction was complete by the time the mixture reached reflux. Aftercooling to ambient temperature, the solution was washed with saturatedNaHCO₃. The organic layer was stripped under vacuum. The residue wasmixed with 1-2 mL of ether and the solution was removed with a pipette,leaving the product which was dried under vacuum.

1.28 Preparation of 2,4,6-trimethyl-pyridine 1-oxide

In a 500 mL round bottom flask equipped with a magnetic stirrer andthermometer were added 36.7 g (164 mmol) of 77% 3-chloroperbenzoic acid(Aldrich) and 200 mL of methylene chloride. This slurry was cooled to 5°C. and a solution of 16.6 g (137 mmol) of collidine (Aldrich) in 50 mLof methylene chloride was added over 30 min while maintaining thetemperature at 5-10° C. The mixture was then allowed to warm to roomtemperature over 1 hr and then stirred overnight. The crude reactionmixture was transferred slowly to a beaker containing 200 g of basicalumina, which resulted in a slight warming of the mixture. The mixturewas stirred and filtered and the alumina was mixed with 300 mL of 2:1CHCl₃:CH₃OH. The solvent was removed on a rotary evaporator at roomtemperature. The alumina was washed with ether and the solvent wasremoved to yield 24.7 g of a clear liquid, which solidified to give awhite, waxy solid. This yield was slightly high due to the presence ofsome salt. TLC (silica gel developed with methanol) showed a singlemajor spot (Rf=0.45) along with a minor spot (Rf=0.55). The major spotwas the desired N-oxide. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 6.96 (s), 2.519(s) and 2.28 (s). The minor spot corresponded to the startingcollidine. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 6.78 (s), 2.48 (s) and 2.26(s).

1.29 Preparation of (4,6-dimethyl-pyridin-2-yl)-methanol

Under an atmosphere of nitrogen, 18.1 g (137 mmol) of the collidineN-oxide was dissolved in 200 mL of methylene chloride dried overmolecular sieves. The mixture was cooled to 5° C. Trifluoroaceticanhydride (71.9 g, 49.8 ml, 343 mmol) was added drop-wise in portions tomaintain the reaction mixture at 5-10° C. After addition of thetrifluoroacetic anhydride, the mixture was allowed to warm to roomtemperature and then stirred at room temperature overnight. SubsequentTLC (reverse phase, methanol/water, 7:3) showed the absence of thestarting N-oxide. The solvent was removed to yield the acetate productas a yellow, waxy solid. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 2.5 (s, 3H),2.8 (s, 3H), 5.65 (s, 2H), and 7.45 (m, 2H). The mixture was cooled inan ice bath and 100 mL of a 10% solution of KOH in methanol was added.The pH of the solution was checked, and if the solution was not basic,additional KOH was added to make the solution basic. The solution wasthen stirred at 10-15° C. for 30 min and then stirred at roomtemperature for 6 hours. The solvent was removed to yield 7.4 g of ayellow-brown syrup. If desired, the alcohol could be purified by carefulchromatography, using silica gel and eluting with ethylacetate/chloroform (4:1). The alcohol was isolated as a pale, yellowoil. TLC: Rf is 0.55 in silica gel, developed with methanol/ethylacetate, 1:1. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 6.67 (s, 2H), 4.67 (s,2H), 2.50 (s, 3H) and 2.31 (s, 3H). In most cases, the crude alcohol wassufficiently pure to be used for the subsequent oxidation reaction.

1.30 Preparation of 4,6-dimethyl-2-pyridinecarboxylic acid

4,6-Dimethyl-2-pyridinemethanol (7.5 g, 29.2 mmol) was added to 100 mLof water and stirred at 0-5° C. A solution of 5.3 g (32.1 mmol) ofpotassium permanganate in 100 mL of water was added portion-wise over 30min while maintaining the temperature at 5-10° C. This resulted in theformation of a black solid. The mixture was stirred at 5-10° C. for anadditional 30 min and then allowed to stir at room temperature for 30min. The mixture was filtered and the manganese dioxide washed withmethanol. The methanol washings were combined with the water extractsand the solvent was removed. The resulting tan solid was redissolved inwater and washed with chloroform. The water layer was separated and thewater removed to yield 5.4 g of 4,6-dimethyl-2-pyridinecarboxylic acid.The product was characterized by HPLC/MS.

1.31 Preparation of pyrazine-2-carboxylic acidN-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide (RG-115550)

Into a 20 mL vial, containing a stirred mixture of 0.120 g (0.003 mol)of NaH in 6 mL of DMF, was slowly added 0.238 g (0.001 mol) of2-ethyl-3-methoxy-benzoic acid N′-tert-butyl-hydrazide. The reaction wasstirred for 1 hr at room temperature. 0.278 g (0.001 mol) ofpentafluorophenyl ester pyrazine-2-carboxylic acid pentafluorophenylester in 2 mL of DMF was slowly added. The reaction was stirred for 24hours. The reaction was washed out with ethyl acetate into a separatoryfunnel containing 100 mL of water and 100 mL of ethyl ether. Thereaction mixture was shaken and the organic phase was dried over MgSO₄and concentrated to dryness to yield pyrazine-2-carboxylic acidN-tert-butyl-N′-(2-ethyl-37-methoxy-benzoyl)-hydrazide: ¹H NMR (CDCl₃,300 MHz) δ (ppm): (note—a 1:3 reaction ratio of hydrazide to NaH gavehighest yield of product (100%), lesser ratios, such as 1:2, onlyyielded 70%; DMF was a better solvent than DMSO). The progress of thereaction was monitored by following the intensity of —OCH₃ signals in ¹HNMR. ¹H NMR (300 MHz, CDCl₃) δ ppm: 9.05 (1H), 8.6 (s, 1H), 8.45 (s,1H), 8.4 (s, 1H), 7.1 (t, 1H), 6.9 (d, 1H), 6.45 (d, 1H), 3.8 (s, 3H),2.4 (m, 1H), 1.95 (m, 1H), 1.62 (s, 9H), 0.95 (t, 3H).Pyrazine-2-carboxylic acid pentafluorophenyl ester: ¹H NMR (300 MHz,CDCl₃) δ ppm: 9.49 (s, 1H), 8.95 (d, 1H), 8.87 (d, 1H).

TABLE 1 Optimization of the preparation of RG-115550

base/ solvent Temp Time yield comment NaH (1 eq) 20 16  40 DMF NaH (2eq) 20 16 65-70 DMF NaH (3 eq) 20 16 100 Pentafluorphenyl ester free ofDMF DCC-derived urea NaH (2 eq) 20 16  45 DMSO

TABLE 2 Preparation of heterocyclic diacylhydrazines by thepentafluorophenyl ester method.

R NMR yield ¹H NMR (300 MHz, CDCl₃)

ca. 30% Diacylhydrazine: 9.1 (s, 1H), 8.4 (d, 1H), 7.85 (d, 1H), 7.1 (t,1H), 6.9 (d, 1H), 6.3 (d, 1H), 4.0 (s, 3H), 3.82 (s, 3H), 2.4 (m 1H),2.1 (m 1H), (s, 9H), 0.95 (t, 3H) R-pentafluorophenyl ester: 9.45 (s,1H), 8.6 (d, 1H), 8.4 (d, 1H), 4.04 (s, 3H)

ca. 20% Diacylhydrazine: Isomer 1: 9.4 (br, 1H), 7.95 (s, 1H), 7.7 (s,1H), 7.3 (m, 10H), 7.1 (m, 5H), 6.85 (t, 1H), 6.73 (d, 1H), 6.5 (d, 1H),6.05 (d, 1H), 3.75 (s, 3H), 2.1 (m, 1H), 1.9 (m, 1H), 1.57 (s, 9H), 0.8(t, 3H). Isomer 2: 8.6 (br, 1H), 7.9 (s, 1H), 7.8 (s, 1H), 7.3 (m, 10H),7.1 (m, 5H), 6.9 (t, 1H), 6.75 (d, 1H), 6.5 (d, 1H), 6.05 (d, 1H), 3.75(s, 3H), 2.1 (m, 1H), 1.9 (m, 1H), 1.57 (s, 9H), 0.85 (t, 3H)R-pentafluorophenyl ester: 8.15 (s, 1H), 8.05 (d, 1H), 7.9 (d, 1H),7.1-7.5 (m, 15H)

100% Diacylhydrazine: 7.75 (s, 1H), 7.55 (d, 1H), 7.35 (d, 1H), 7.3 (m,1H), 7.15 (t, 1H), 7.0 (t, 1H), 6.8 (d, 1H), 6.6 (s, 1H), 6.4 (d, 1H),3.9 (s, 3H), 3.75 (s, 3H), 2.2 (m, 1H), 1.9 (m, 1H), 1.62 (s, 9H), 0.85(t, 3H) R-pentafluorophenyl ester: 7.75 (d, 1H), 7.65 (s, 1H), 7.5 (brs, 2H), 7.2 (m, 1H), 4.09 (s, 3H)

100% Diacylhydrazine: 7.85 (s, 1H), 7.65 (s, 1H), 7.5 (m, 3H), 7.4 (m,2H), 7.15 (t, 1H), 6.9 (d, 1H), 6.65 (d, 1H), 3.81 (s, 3H), 2.6 (m, 1H),2.5 (s, 3H), 2.2 (m, 1H), 1.6 (s, 9H), 1.1 (t, 3H) R-pentafluorophenylester: 8.25 (s, 1H), 7.6 (m, 3H), 7.5 (m, 2H), 2.62 (s, 3H)

100% Diacylhydrazine: 8.5 (s, 1H), 7.95 (s, 1H [NH]), 7.1 (t, 1H), 6.87(d, 1H), 6.3 (d, 1H), 3.85 (s, 3H), 2.55 (s, 3H), 2.5 (m, 1H), 2.3 (m,1H), 1.64 (s, 9H), 1.05 (t, 3H). R-pentafluorophenyl ester: 8.8 (s, 1H),2.62 (s, 3H) Procedure for pentafluorophenyl ester formation:Heterocyclic carboxylic acid and pentafluorphenol are dissolved inanhydrous dioxane, ethyl acetate, dimethoxyethane, or THF under an N₂atmosphere. One equivalent of dicyclohexylcarbodiimide (DCC) is added.The reaction is stirred at room temperature overnight. A trace of wateris then added to quench any remaining DCC. The DCC-derived urea (DCU) isremoved by filtration on Celite, the filtrate is washed with diluteNaHCO₃ to remove remaining pentafluorophenol, and the filtrate isevaporated to dryness. The product is purified by trituration orchromatography on silica gel. It is thought that the level of trace DCCor urea in the pentafluorophenyl ester may be critically detrimental tothe success of the NaH amide coupling reaction.1.32 Preparation of 1H-Indazole-3-carboxylic acidN-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide (RG-115723)

Into a 10 mL round bottom flask, was added 0.328 g (0.001 mol) ofpentafluorophenyl ester of indazole-3-carboxylic acid, 0.244 g (0.001mol) of triphenylmethane, 0.227 g (0.001 mol) of 2,3-dichloro,5,6-dicyano, 1,4-benzoquinone and 4 mL of dry toluene. The reactionmixture was refluxed for 7-8 hours. The reaction mixture was washed outwith ethyl acetate (60 mL) into a separatory funnel and extracted withwater (20 mL). The organic phase was dried and concentrated to yield0.45 g of 1-trityl-1H-indazole-3-carboxylic acid pentafluorophenylester. NMR indicated the presence of the product, but TLC also showedthe presence of the starting pentafluorophenyl ester (Rf 57) and product(Rf 70). The product was purified by column chromatography on silicagel, eluted with 5% ethyl acetate/hexane to yield 0.30 g of pureproduct. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 8.2 (d, 1H), 7.4-7.0 (m, 17H),6.52 (d, 1H).

0.17 g (0.16 mmol) of 1-trityl-1H-indazole-3-carboxylic acidpentafluorophenyl ester in 3 mL of CH₂Cl₂ was added to a 100 mL flaskwith 20 mL of CH₂Cl₂, containing 2% TFA and 1% H₂O. The reaction wasstirred at room temperature for 90 min. TLC indicated 50% reaction. Anadditional 15 mL of the TFA —H₂O—CH₂Cl₂ was added and stirring continuedfor 1 hr. The reaction was transferred to a separatory funnel and washedwith ca. 0.5 M K₂CO₃H₂O. The CH₂Cl₂ phase was dried and concentrated togive crude 1H-indazole-3-carboxylic acidN-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide (0.15 g). TLC:product, Rf 0.33; starting material, Rf=0.68. The product was purifiedby column chromatography, eluting with 45% ethyl acetate/hexane. ¹H NMR(CDCl₃, 300 MHz) δ (ppm): 8.6 (s, 1H), 8.2 (s, 1H), 7.4-7.2 (m, 3H), 7.1(t, 1H), 6.9 (d, 1H), 6.7 (d, 1H), 3.80 (s, 3H), 2.4 (m, 1H), 2.0 (m,1H), 1.652 (s, 9H), 0.85 (t, 3H).

1.33 Preparation of 3H-benzoimidazole-5-carboxylic acidN-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide (RG-115718)

About 120 mg of 3-trityl-3H-benzoimidazole-5-carboxylic acidN-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide were dissolved in35 mL of CH₃OH and added into a hydrogenation bottle, together with 2drops of glacial acid and 0.20 g Pd/C. Hydrogenation was conducted byshaking the bottle for 6 hours and then remaining under H2 pressure for16 hours. The Pd/C was removed by filtration and the methanol removed byan evaporator. The residue was stirred with CH₂Cl₂ and the CH₂Cl₂ wasdecanted. Evaporation of the CH₂Cl₂ yielded a solid product identifiedby NMR, as triphenylmethane. The residue was stirred with dilute KOH/H₂Oand extracted with ethyl acetate. The ethyl acetate was dried andconcentrated on a rotary evaporator to yield the product3H-benzoimidazole-5-carboxylic acidN-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide. NMR analysis ofthe product indicated the absorbances for the methoxy and t-Butyl groupsoccurred at 3.69 and 1.61 vs. 3.77 and 1.59 for the starting material.

1.34 Preparation ofN′-2-Ethyl-3-methoxybenzoyl-N-t-butyl-N—(N-methylindole-2-carbonyl)hydrazide

N′-2-Ethyl-3-methoxybenzoyl-N-t-butyl hydrazide (150 mg, 0.6 mmol) wasdissolved in 2 mL of DMF. Potassium t-butoxide (80 mg, 0.7 mmol) wasadded and magnetically stirred for about 5 min.N-Methylindole-2-carboxylic acid pentafluorophenyl ester was added andthe mixture was heated to 100° C. After 3 hours the reaction wascomplete as indicated by TLC. The mixture was cooled to ambienttemperature and poured into 10 mL of water. Two extractions withmethylene chloride were combined and evaporated. The residue was mixedwith about 2 mL of 1:1 ethyl ether: hexane. The mother liquors wereremoved by pipette and the residue dried under vacuum. The product was atan solid weighing 140 mg. LC MS analysis confirmed the structure andestimated the purity (UV detection) at 91%. (Yield=52%).

1.35 Preparation of 2,6-dimethoxy-nicotinic acidN-tert-butyl-N′-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide(RG-115517)

2.0 g (10.92 mmol) of 2,6-dimethoxyisonicotinic acid was dissolved in100 mL of toluene and then 1 drop of dimethyl formamide was added. 1.55g (13.1 mmol, 0.98 mL) of thionyl chloride was added and the solutionwas refluxed for 4 hours. The toluene and excess thionyl chloride wereremoved under vacuum and 2,6-dimethoxyisonicotinoyl chloride was usedwithout further purification.

In a 1 oz vial, with a stirbar, 1 mL of 1 M K₂CO₃ was added. 0.250 g(1.2 mmol) of 5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acidN′-tert-butyl-hydrazide was dissolved in 2 mL of methylene chloride andadded to the aqueous solution. 2,6-dimethyl-isonicotinoyl chloride wasthen added and the mixture was allowed to stir at room temperatureovernight. The aqueous layer was removed and the organic layer waswashed twice with 2 mL of a 1 M K₂CO₃ solution followed by 2 mL ofwater. The water layer was removed and the organic layer was dried overMgSO₄. The organic layer was filtered and then removed. The product,2,6-dimethoxy-nicotinic acidN-tert-butyl-N′-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide,was purified by trituration with 1:1 ether:hexane or chromatography. ¹HNMR (CDCl₃, 300 MHz) δ (ppm): 1.6 (s, 9H), 1.9 (s, 3H), 3.9 (s s, 6H),4.2 (m, 4H), 6.2 (m, 1H), 6.7 (d, 1H), 7.7 (d, 1H), 8.3 (m, 1H).

1.36 Preparation of 4-Hydroxy-3,5-dimethoxy-benzoic acidN-tert-butyl-N′-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide(RG-115009)

4-Acetoxy-3,5-dimethoxy-benzoic acid (1.45 g) was heated with thionylchloride (0.86 g) in 3 mL of dimethoxyethane. After 1.5 hours themixture was stripped under vacuum leaving 1.60 g of4-acetoxy-3,5-dimethoxy-benzoyl chloride as an oil. ¹H NMR (CDCl₃, 300MHz) δ (ppm): 2.26 (s, 3H), 3.89 (s, 9H), 7.34 (s, 2H).

4-Acetoxy-3,5-dimethoxy-benzoyl chloride (250 mg) and5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acidN′-tert-butyl-hydrazide (204 mg) were dissolved in 3 mL ofdichloromethane and stirred at ambient temperature with 1.5 mL of a 1 Maqueous sodium carbonate solution. After two hours the phases wereseparated and the organic phase was evaporated. The solid residue waswashed with 1:1 ether:hexane leaving 360 mg of acetic acid4-[N-tert-butyl-N′-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazinocarbonyl]-2,6-dimethoxy-phenylester. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 1.60 (m, 15H), 2.06 (s, 3H),2.32 (s, 3H), 3.77 (s, 6H), 4.2 (m, 4H), 6.08 (d, 1H), 6.63 (d, 1H),6.74 (s, 2H).

Acetic acid4-[N-tert-butyl-N′-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazinocarbonyl]-2,6-dimethoxy-phenylester (300 mg) was dissolved in methanol with 28% aqueous ammonia (750mg). The mixture was stirred at ambient temperature over the weekend.The precipitate was filtered to provide 110 mg of white solid4-hydroxy-3,5-dimethoxy-benzoic acidN-tert-butyl-N′-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide.¹H NMR (CDCl₃, 300 MHz) δ (ppm): 1.59 (s, 15H), 2.02 (s, 3H), 3.85 (s,6H), 4.21 (m, 4H), 5.6-5.7 (broad s, 1H), 6.22 (d, 1H), 6.58 (d, 1H),6.81 (s, 2H).

1.37 Preparation of Compounds RG-115613 and RG-115429

Into a 100 mL round bottom flask containing 2.50 g (10 mmol) of2-ethyl-3-methoxy-benzoyl-N′-tert-butyl-hydrazide was added 15 mL ofmethylene chloride, 2.60 g (10.5 mmol) of 3-bromomethyl-5-methylbenzoylchloride in 5 mL of methylene chloride and a solution of 2.76 g (20mmol) of potassium carbonate in 15 mL of water. The reaction mixture wasstirred overnight at room temperature, then diluted with 20 mL ofmethylene chloride and transferred to a separatory funnel. The methylenechloride layer was separated and dried, and the solvent was removed invacuo. The crude product was purified by column chromatography to yield4.01 g ofN-(3-bromomethyl-5-methyl-benzoyl)-N-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)hydrazide (87% yield). ¹H NMR (CDCL₃, 300 MHz) δ (ppm): 7.41 (s, 1H),7.1 (m, 3H), 7.02 (t, 1H), 6082 (d, 1H), 6.08 (d, 1H), 4.41 (s, 2H),3.78 (s, 3H), 2.4 (m, 1H), 2.31 (s, 3H), 2.25 (m, 1H), 1.60 (s, 9H),1.01 (t, 3H).

To 4.00 g (8.68 mmol) ofN-(3-bromomethyl-5-methyl-benzoyl)-N-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)hydrazide, contained in a 250 mL round bottom flask, were added 40 mL ofdioxane, 40 mL of water, and 4.34 g of calcium carbonate. The reactionflask was placed into an 85° C. oil bath and the reaction was stirredand heated for 18 hours. The reaction mixture was cooled, transferred toa larger flask with ethyl acetate and most of the dioxane wasevaporated. The reaction mixture was shaken with about 100 mL of ethylacetate and filtered. The ethyl acetate layer was separated and theaqueous layer extracted twice with ethyl acetate. Ethyl acetate extractwas dried and evaporated to yield 2.07 g ofN-(3-hydroxymethyl-5-methyl-benzoyl)-N-tert-butyl-N′-(2-ethyl-3-methoxy-benzyl)hydrazide (60% yield). ¹H NMR (300 MHz, CDCl₃) δ (ppm): 7.78 (s, 1H),7.1-7.4 (3s, 3H), 6.96 (t, 1H), 6.8 (d, 1H), 6.08 (d, 1H), 4.53 (s, 2H),3.77 (s, 3H), 2.35 (m, 1H), 2.32 (s, 3H), 2.2 (m, 1H), 1.60 (s, 9H),0.96 (t, 3H).

To 2.00 g (5.02 mmole) ofN-(3-hydroxymethyl-5-methyl-benzoyl)-N-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)hydrazideplaced in a 250 mL round bottom flask, were added 100 mL of methylenechloride and 1.16 g of pyridinium chlorochromate. The reaction mixturewas refluxed for about 1 hour, at which time TLC (1:1 ethylacetate:hexane) indicated the formation of the product (Rf=0.5). Thereaction mixture was concentrated to about 20 mL and thenchromatographed on silica gel. Elution with 30-35% ethyl acetate inhexane yielded 1.75 g (88%) 3-formyl-5-methyl-benzoic acidN-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide as a white solid.¹H NMR (300 MHz, CDCl₃) δ (ppm): 9.93 (s, 1H), 7.6-7.8 (3s, 3H), 7.0 (t,1H), 6.82 (d, 1H), 6.19 (d, 1H), 3.77 (s, 3H), 2.42 (s, 3H), 2.3 (m,1H), 2.0 (m, 1H), 1.62 (s, 9H), 0.90 (t, 3H).

To 100 mg (0.25 mmoles) ofN-(3-formyl-5-methyl-benzoyl)-N-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazideplaced in a 20 mL vial, were added 2 mL of methanol, 112 mg ofsemicarbazide hydrochloride, 0.2 g triethylamine and a drop of glacialacetic acid. The reaction mixture was magnetically stirred on a hotplate adjusted to 50° C. for about 3 hours, then at room temperature for48 hours. The solvent was evaporated with a stream of nitrogen and theresulting residue was dissolved in 20 mL of chloroform and extractedwith dilute HCl. The chloroform extract was dried, the solvent wasremoved in vacuo, and the residue was dried in a vacuum oven at 60° C.The residue was cooled and triturated with hexane to yield 81 mg ofsemicarbazide ofN-(3-formyl-5-methyl-benzoyl)-N-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)hydrazide. ¹H NMR (300 MHz, CDCl₃) δ (ppm): 8.9 (broad s, 1H), 8.6(broad s, 1H), 7.3-7.5 (3s, 3H), 7.03 (t, 1H), 6.8 (d, 1H), 6.38 (d,1H), 3.76 (s, 3H), 3.26 (d, 1H), 2.4 (s, 3H), 1.95 (m, 1H), 1.57 (s,9H), 0.95 (t, 3H).

To 100 mg (0.25 mmol) ofN-(3-formyl-5-methyl-benzoyl)-N-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazideplaced in a 20 mL vial, was added 2 mL of methanol, 103 mg of oxamichydrazide hydrochloride, and a drop of glacial acetic acid. The reactionmixture was magnetically stirred on a hot plate adjusted to 50° C. forabout 3 hours, then at room temperature for 48 hours. The solvent wasevaporated with a stream of nitrogen and the resulting residue was driedin a vacuum oven at 60° C. The residue was cooled and triturated withhexane to yield 70 mg of oxamic hydrazone ofN-(3-formyl-5-methyl-benzoyl)-N-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)hydrazide. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.5 (s, 1H), 8.6 (d, 1H),7.9 (d, 1H), 7.55 (s, 1H), 7.5 (s, 1H), 7.3 (s, 1H), 7.0 (t, 1H), 6.95(d, 1H), 3.73 (s, 3H), 2.2 (m, 1H), 2.35 (s, 3H), 1.95 (m, 1H), 1.51 (s,9H), 0.80 (t, 3H).

1.38 Preparation of 3,5-dichloro-4-fluorobenzoic acid

(N-(3-methoxy-2-methylbenzoyl)-N′-(3,5-dichloro-4-fluorobenzoyl)-N′-tert-butylhydrazinecan be prepared according to U.S. Pat. No. 5,530,028. Briefly, theproduct of Example 8 and 3,5-dichloro-4-fluorobenzoyl chloride can beprepared in accordance with Example 9 to yield(N-(3-methoxy-2-methylbenzoyl)-N′-(3,5-dichloro-4-fluorobenzoyl)-N′-tert-butylhydrazine.

3,5-dichloro-4-fluorobenzoyl chloride can be prepared as follows: To around bottom flask with nitrogen purge through a 10% aqueous NaOH trap,was added 3,5-dichloro-4-fluorobenzotriflouride (5.00 g, 21.46 mmol,Aldrich), concentrated sulfuric acid (4.30 g, 42.92 mmol), and finallychlorosulfonic acid (5.15 g, 43.78 mmol). The reaction began to bubbleimmediately. After the bubbling subsided, the mixture was heated to 80°C. for 1 hr, cooled to room temperature, and added cautiously to stirredice water. This was extracted twice with methylene chloride. Thecombined extracts were washed with water and brine, dried over magnesiumsulfate, filtered, and evaporated to give a white solid (2.19 g) of3,5-dichloro-4-fluorobenzoic acid in 49% yield. ¹H-NMR (CD₃COCD₃, 300MHz) δ (ppm): 7.95 (d, 2H).

3,5-dichloro-4-fluorobenzoic acid was refluxed with >1 equivalent ofthionyl chloride and one drop of DMF neat or as a solution in CHCl₃. Thesolvent and volatile by-products were removed in vacuo to provide3,5-dichloro-4-fluorbenzoyl chloride.

1.39 Preparation of 3,5-dimethoxy-4-methyl-2-nitrobenzoic acidN-t-butyl-N′-(5-methyl-benzo-1,4-dioxan-6-carbonyl)-hydrazide(RG-115609)

3,5-Dimethoxy-4-methylbenzoic acid was slurried with 2.7 eq. Of aceticanhydride and 0.05 molar equivalents of concentrated sulfuric acid indichloromethane. After cooling to 10° C., 1.05 eq. Of 70% nitric acidwas added drop-wise while the temperature was maintained below 15° C.After 30 min the mixture was poured into water and extracted twice withethyl acetate. The combined organic extracts were concentrated until athick slurry was present. The slurry was filtered and the solid washedwith ice-cold dichloromethane. Further concentration of the motherliquors gave a second crop. The total yield of3,5-dimethoxy-4-methyl-2-nitrobenzoic acid was about 80%. ¹H NMR:(acetone-d6, 300 MHz) δ (ppm): 7.34 (s, 1H), 4.00 (s, 3H), 3.85 (s, 3H),2.23 (s, 3H).

3,5-Dimethoxy-4-methyl-2-nitrobenzoic acid was stirred with 1.1 eq. Ofthionyl chloride at ambient temperature in dimethoxyethane until thereaction was complete. The solvent and excess thionyl chloride weredistilled at atmospheric pressure and the residue dissolved indichloromethane. This solution was added to a mixture of 1 M aqueouspotassium carbonate and 5-methylbenzo-1,4-dioxan-6-benzoicacid-N′-t-butyl-hydrazide in dichloromethane. After 3 hr, water anddichloromethane were added. The organic phase was removed and strippedunder vacuum. The residue was triturated with 1:1 (wt:wt) ether: hexaneto provide 3,5-dimethoxy-4-methyl-2-nitrobenzoic acidN-t-butyl-N′-(5-methyl-1benzo-1,4-dioxan-6-carbonyl)-hydrazide (ca 94%yield). TLC (1:1 ethyl acetate:hexane) indicated one spot, Rf 0.53. ¹HNMR: (CDCl₃, 300 MHz) δ (ppm): 7.84 (s, 1H), 7.02 (t, 1H), 6.86 (d, 1H),6.82 (s, 1H), 6.11 (s, 1H), 3.90 (m, 4H), 3.79 (s, 6H), 2.16 (s, 3H),1.60 (s, 9H).

1.40 Preparation of 3,5-dimethyl-benzoic acidN′-(2,3-dimethyl-benzoyl)-N-(1-ethyl-2,2-dimethyl-propyl)-hydrazide(RG-103309)

T-butylcarbazate (35.15 g, 266 mmol) and 200 mL of CH₂Cl₂ were added toa round bottom flask. Potassium carbonate (55.2 g, 0.4 moles) dissolvedin 350 mL of water was added to the flask, and the mixture was stirredfor 15 minutes with ice chilling. 2,3 dimethylbenzoyl chloride (44.9 g,266 mmol) in ca. 200 mL of CH₂Cl₂ was added drop-wise from a 500 mLseparatory funnel over 30 minutes. The reaction was allowed to stirovernight and then the reaction mixture was poured into a 1 L separatoryfunnel and the CH₂Cl₂ phase was separated. Then ca. 150 mL of water wasadded, and the mixture was extracted twice with 150 mL of CHCl₃. Thecombined organic phase was back-extracted with 100 mL of water, thenwith 1N HCl (250 mL), to remove the hydrazide. The organic phase wasdried, stirred with charcoal, and the solvent removed in vacuo to yielda light tan solid (71.5 g) ofN′-(2,3-dimethyl-benzoyl)-hydrazinecarboxylic acid, tert-butyl ester. ¹HNMR (300 MHz, CHCl₃) δ (ppm): 7.7 (br, 1H), 7.22 (m, 2H), 7.1 (t, 1H),7.85 (br, 1H), 2.35 (s, 3H), 2.3 (s, 3H), 1.5 (s, 9H).

N′-(2,3-Dimethyl-benzoyl)-hydrazinecarboxylic acid, tert-butyl ester(70.3 g, 266 moles) was placed in to a 500 mL round bottom flask. Withgentle stirring, 200 mL of trifluoroacetic acid (296 g, 2.6 moles) wasslowly added, resulting in a vigorous evolution of gas. The reactionmixture was then stirred at room temperature for 2 hours. Water (ca. 100mL) was then added slowly to the mixture. The mixture was slowly addedto 1 L of a cold 2 M K₂CO₃ solution, contained in a 2 L beaker, whilestirring slowly (evolution of gas). About 200 mL of a 10% NaOH solutionand 250 mL of CH₂Cl₂ were added. The reaction mixture was transferred toa large separatory funnel and gently shaken (gas evolution). The aqueousphase was extracted with CHCl₃ and the extracts dried and evaporated toyield a white solid, which was dried in a 50° C. vacuum oven to yield31.72 g (73% yield) of 2,3-dimethyl-benzoic acid hydrazide. ¹H NMR(CDCl₃, 300 MHz) δ (ppm): 7-7.3 (m, 4H), 4.00 (br s, 2H), 2.271 (s, 6H).

In a 200 mL round bottom flask, 7.90 g (48 mmol) of 2,3-dimethyl-benzoicacid hydrazide was dissolved in 60 mL of methanol and 3 drops of glacialacetic acid were then added. To the reaction mixture was added 6.00 g(52.6 mmol) of 2,2-diemthylpentan-3-one, and the reaction was stirred atroom temperature for 24 hours. The product hydrazone was not isolated,but subjected directly to reduction. Glacial acetic acid (10 mL) andsodium cyanoborohydride (3.2 g, 50.95 mmol) were added to the reactionmixture, which was then stirred at room temperature for 24 hours. About50 mL of 10% aqueous NaOH solution was added and most of the CH₃OH wasremoved on a rotary evaporator. The reaction was diluted with water (100mL) and the product was extracted with CH₂Cl₂. The organic extract wasdried and evaporated to yield 11.87 g (94%) of 2,3-dimethyl-benzoic acidN′-(1-ethyl-2,2-dimethyl-propyl)-hydrazide. ¹H NMR (500 MHz, CDCl₃) δ(ππμ): 7.0-7.3 (m, 4H), 2.32 (s, 3H), 2.296 (s, 3H), 1.7 (m, 1H), 1.3(m, 1H), 1.16 (t, 3H, 0.979 (s, 9H). TLC: Rf=0.57 (1:1 ethylacetate:hexane), indicated >90% purity. Further purification can beachieved by silica gel chromatography and elution of product with 20%ethyl acetate in hexanes.

2,3-dimethyl-benzoic acid N′-(1-ethyl-2,2-dimethyl-propyl)-hydrazide(0.59 g, 2.25 mmol) was dissolved in 15 mL of CH₂Cl₂ in a smallround-bottom flask. Aqueous K₂CO₃ solution (0.70 g in 150 mL of H₂O) wasadded. 3,5-dimethylbenzoyl chloride (0.45 g, 2.7 mmol) dissolved in 10mL of CH₂Cl₂ was added and the reaction mixture was stirred at roomtemperature for 24 hours. The reaction mixture was transferred to aseparatory funnel and extracted with CH₂Cl₂. (In another experiment,this was washed with weak NaOH to get rid of excess acid and acidchloride). The extract was dried and evaporated to give about 1 g of awhite solid, which was purified by silica gel chromatography. Elutionwith 15% ethyl acetate in hexane yielded pure product of3,5-dimethyl-benzoic acidN′-(2,3-dimethyl-benzoyl)-N-(1-ethyl-2,2-dimethyl-propyl)-hydrazide(0.62 g, 70%). ¹H NMR (500 MHz, CDCl₃) 6=(ppm): 6.95-7.4 (m, 7H), 4.61(m, 1H), 2.2-2.4 (multiple s, 9H), 1.81 (s, 3H), 1.6-1.8 (m, 2H), 1.3(br t, 3H), 1.08 (multiple br s, 9H).

1.41 Preparation of 3,5-dimethyl-benzoic acidN-(1-ethyl-2,2-dimethyl-propyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide(RG-115819)

10.34 g (57.5 mmol) of 3-methoxy-2-methyl-benzoic acid hydrazide (lotCPO 10925) was dissolved in 100 mL of methanol and stirred. 9.80 g (86.3mmol) of 2,2-dimethyl-3-pentanone was added followed by 3 drops ofglacial acetic acid. The mixture was allowed to stir for 48 hours. Thecrude mixture was then brought to pH=3. 3.84 g (61.2 mmol) of NaCNBH₃was then added. The slurry was allowed to stir at room temperatureovernight, and then the methanol was removed. 100 mL of 10% NaOH and 100mL methylene chloride were added. The mixture was shaken and themethylene chloride layer removed. The aqueous layer was washed twicewith 75 mL of methylene chloride. The methylene chloride layers werecombined and dried. Removal of the solvent yielded the desired productas a pale, yellow liquid. The product could also be obtained byreduction of the hydrazide with 10% Pd on carbon. Purification wasaccomplished by column chromatography on silica gel, eluting with 3:2hexane:ether. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 1.0 (s, 9H), 1.1 (t, 3H)1.2 (m, 1H), 1.5 (m 2H), 2.1 (s, 3H), 3.8, (s, 3H), 4.9 (br s, 1H), 6.6(d, 1H), 7.0-7.2 (m, 2H). TLC: Rf=0.45 (1:1 hexane:ether).

6.2 g (22.1 mmol) of 3-methoxy-2-methyl-benzoic acid(1-ethyl-2,2-dimethyl-propyl)-hydrazide was dissolved in 35 mL of ethylacetate and cooled to 0° C. 74 mL of a 1N aqueous K₂CO₃ was added andthe mixtures stirred. 5.6 g (33.6 mmol) of 3,5-dimethylbenzoyl chloridewas dissolved in 40 mL ethyl acetate and this solution was added to thehydrazide mixture over 15 min. The mixture was allowed to warm to roomtemperature and stirred overnight. The aqueous layer was then removedand the organic layer was washed with 75 mL of a 1N aqueous K₂CO₃solution and then with 100 mL of water. The water layer was removed andthe organic layer was dried and removed to yield an off-white solid.This material was triturated three times with 25 mL of 1:1 hexane:etherto yield the final product in 98.7% purity. ¹H NMR (CDCl₃, 300 MHz) δ(ppm): 0.7-1.5 (m, 15H), 2.1 (s, 9H), 3.7 (s, 3H), 6.8-7.1, (m, 6H);TLC: Rf=0.62 (1:1 hexane:ether).

1.42 Preparation of 3,5-dimethoxy-4-methyl-benzoic acidN-(1-ethyl-2,2-dimethyl-propyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide(RG-115820)

Into a 20 mL vial was added 161 mg (0.75 mmol) of 3,5 dimethoxy,4-methyl benzoyl chloride, a 5 mL solution of 3-methoxy-2-methyl-benzoicacid N′-(1-ethyl-2,2-dimethyl-propyl)-hydrazide, and 1.5 mL of aqueous25% K₂CO₃. The reaction mixture was stirred at room temperature for 24hours. The reaction mixture was transferred to a separatory funnel withCH₂Cl₂ and shaken with dilute aqueous NaHCO₃. The organic phase wasdried, concentrated and chromatographed on silica. 100 mg of pureproduct, 3,5-dimethoxy-4-methyl-benzoic acidN-(1-ethyl-2,2-dimethyl-propyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide-,was eluted with 25% ethyl acetate/hexane. TLC: Rf=0.54 (1:1 ethylacetate:hexane); ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 6.6-7.2 (m, 4H), 6.25(d, 1H), 4.6 (d, 1H), 3.8-3.95 (br s, 9H), 2.1 (br s, 3H), 1.9 (s, 3H),1.6 (br, 2H), 1.3 (m, 3H), 0.9-1.2 (br d, 9H).

1.43 Preparation of 2,2-dimethyl-heptan-3-ol

20 g (0.232 mol) of pivaldehyde dissolved in 600 mL of THF was added toa 2 L 3-neck round bottom flask equipped with a magnetic stir bar,thermometer and rubber stopper. The vessel was maintained under N₂. Thereaction mixture was cooled to −65° C. in a dry ice/acetone bath. 112 mL(0.279 mol) of a 2.5 M BuLi solution in hexane was slowly added in 5 mLportions with a 20 mL glass syringe, maintaining the temperature below−55° C. The reaction was stirred at 60° C. for one hour, then allowed towarm to −5° C. over one hour. The reaction was cooled again to 60° C.and slowly quenched with NH₄Cl/H₂O solution, maintaining the temperaturebelow −50° C. 100 mL of water were added and the reaction was allowed towarm to room temperature. The THF was removed on a rotary evaporatorwith a bath temperature of 25° C. until an oil was observed. The productwas extracted with ethyl ether, and the ether was dried and evaporatedcarefully to yield 31.0 g of 2,2-dimethyl-heptan-3-ol that was useddirectly in a subsequent oxidation reaction. ¹H NMR (CDCl₃, 500 MHz) δ(ppm): 3.2 (m, 1H), 1.2-1.7 (m, 3H), 0.93 (m, 3H), 0.89 (s, 9H).

1.44 Preparation of 2,2-dimethyl-heptan-3-one

2,2-Dimethyl-heptan-3-ol (0.23 mol) was dissolved in 350 mL of CH₂Cl₂ ina 500 mL round bottom flask with a magnetic stirbar. The flask waspartially cooled with ice. 76.6 g (0.355 mol) of pyridiniumchlorochromate was added, while vigorously stirring. The reaction turnedblack and warmed up slightly. The reaction mixture was stirred at roomtemperature for 24 hours. The solution was decanted away from the blacksludge, which was rinsed with hexane. The organic extracts were combinedand chromatographed directly on silica gel. (Note: only silica has beenfound to trap and remove the reduced non-reacted chromium compounds).The product, 2,2-dimethyl-heptan-3-one, eluted with CH₂Cl₂/hexane and ina subsequent 10% ethyl acetate/hexane fraction to yield 29.19 g ofproduct at 88% yield. ¹H NMR (CDCl₃, 500 MHz) δ (ppm): 2.48 (t, 2H),1.54 (m, 2H), 1.28 (m, 2H), 1.13 (s, 9H), 0.90 (m, 3H).

1.45 Preparation of 3-methoxy-2-methyl-benzoic acidN′-(1-tert-butyl-pentyl)-hydrazide

2.84 g (20 mmol) of 2,2-dimethyl-heptan-3-one, 3.60 g (20 mmol) of2-methyl, 3-methoxy benzoic acid hydrazide, 20 drops of glacial aceticacid and 40 mL of 100% ethyl alcohol were refluxed for 4 hours and thenstirred at room temperature for 24 hours. TLC indicated only a 35%reaction. Accordingly, the reaction mixture was refluxed for anadditional 6 hours. TLC indicated ca. 80% reaction (TLC Rf=0.57,starting hydrazide, Rf=0.08, 1:1 ethyl acetate:hexane). To the reactionmixture was added, 3.5 mL of glacial acetic acid and 1.89 g (30 mmol) ofNaCNBH₃. The mixture was stirred at room temperature for 2 hours andrefluxed for 1 hour. 50 mL of water was added and 15% NaOH was addeduntil the reaction mixture was basic. Most of the alcohol was removed ona rotary evaporator and the product was extracted with CHCl₃, to yield4.28 g of crude material. TLC indicated the product hydrazide at Rf 0.54(1:1 ethyl acetate:hexane). Purification by gradient chromatography onsilica yielded 3.03 g of product, which eluted in a 25-40% ethylacetate/hexane fraction. Drying in a vacuum oven at 55° C. eliminatedvolatile materials, yielding 2.69 g of 3-methoxy-2-methyl-benzoic acidN′-(1-tert-butyl-pentyl)-hydrazide. ¹H NMR (CDCl₃, 500 MHz) δ (ppm): 7.2(t, 1H), 7.05 (br, 1H[NH]), 6.9 (m, 2H), 4.9 (br, 1H), 3.84 (s, 3H), 2.5(m, 1H), 2.3 (s, 3H), 1.2-1.8 (m, 6H), 0.97 (s, 9H), 0.92 (t, 3H).

17.8 5 g (90.98 mmol) of 4-methoxybenzyl carbazate was dissolved in 50mL of CH₂Cl₂ in a 250 mL flask and then cooled in ice water. 21.42 g(155 mmol) of potassium carbonate dissolved in 80 mL of water was added.While the reaction mixture was being stirred in the ice bath, 17.0 g(79.95 mmol) of 5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonylchloride in 60 mL of CH₂Cl₂ were slowly added. The reaction mixture wasstirred at room temperature overnight and then transferred to aseparatory funnel with 200 mL of CH₂Cl₂ and 200 mL of H₂O. Aftershaking, a floating white precipitate was filtered off, washed withwater, dried in a vacuum oven to give 29.1 g ofN′-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)hydrazinecarboxylicacid 4-methoxy-benzyl ester. The CH₂Cl₂ solution was dried andconcentrated to give 5.19 g of a residue consisting of the original acidchloride, 4-methoxybenzyl carbazate, and some product. TLC Rf=0.38(streak 1:1 ethyl acetate:hexane). ¹H NMR (CDCl₃, 300 MHz) δ (ppm):7.4-6.7 (m, 6H), 5.139 (s 2H), 4.279 (s 4H), 3.81 (s, 3H), 2.303 (s,3H).

In a 500 mL volume flask, was combined 18.6 g (0.0499 moles) ofN′-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazinecarboxylicacid 4-methoxy-benzyl ester, 72 mL of concentrated HCl, and 108 mL ofdioxane. The flask was placed into an 80° C. oil bath and mechanicallystirred for 2 hours. The reaction mixture was cooled with ice water,then poured onto ice water and transferred to a separatory funnel. Thereaction mixture —H₂O solution was then extracted twice with 150 mL ofCH₂Cl₂ to remove the acids and neutrals (the starting material). Theaqueous phase was made basic (pH 12) with a 20% NaOH solution andextracted 4 times with 150 mL of ethyl acetate. The ethyl acetateextract was dried over MgSO₄ and concentrated to yield 4.5 g of5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid hydrazide. ¹HNMR (CDCL₃, 300 MHz) δ (ppm): 7.0 (s, 1H), 6.85 (d, 1H), 6.74 (d, 1H),4.28 (m, 4H), 2.781 (s, 3H).

1.46 Preparation of 5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylicacid N′-(1-ethyl-2,2-dimethyl-propyl)-hydrazide

0.86 g (4.1 mmol) of -methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylicacid hydrazide and 1.14 g (10 mmol) of 2,2 dimethyl pentan-3-one, 30 mLof ethyl alcohol and 20 drops of glacial acetic acid were refluxed for 6hours. TLC indicated ca. a 60% conversion to5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid(1-ethyl-2,2-dimethyl-propylidene)-hydrazide (Rf=0.40, 1:1 ethylacetate:hexane). The reaction was cooled, 3 mL of glacial acetic acidfollowed by 0.63 g (10 mmol) of sodium cyanoborohydride were added, andthe reaction was stirred at room temperature for 3 hours. Most of thealcohol was removed on a rotary evaporator. 30 mL of water was added,followed by the addition of 10% NaOH/H₂O until the reaction mixture wasbasic. The mixture was extracted extensively with ethyl acetate. Theethyl acetate extract was dried and evaporated to give 1.2 g of crudematerial. The product was purified by column chromatography on silicagel, eluting with 20-30% ethyl acetate/hexane. About 0.46 g of pure5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acidN′-(1-ethyl-2,2-dimethyl-propyl)-hydrazide was obtained. TLC: Rf=0.46(1:1 ethyl acetate:hexane). ¹H NMR (CDCl₃, 500 MHz) δ (ppm): 7.1 (br,1H[NH]), 6.85 (d, 1H), 6.71 (d, 1H), 4.8 (br, 1H), 4.29 (m, 2H), 4.25(m, 2H), 2.4 (m, 1H), 2.29 (s, 3H), 1.7 (m, 1H), 1.3 (m, 1H), 1.15 (t,3H), 0.98 (s, 9H).

1.47 Preparation of RG-115858, 3,5-dimethyl-benzoic acidN′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-N-(1-ethyl-2,2-dimethyl-propyl)-hydrazide

2.38 g (18 mmol) of t-butyl carbazate were dissolved in 50 mL of CH₂Cl₂in a 250 mL round bottom flask and cooled to 0° C. An aqueous K₂CO₃solution was prepared (4.15 g K₂CO₃/35 mL H₂O) and added to the reactionmixture which was again cooled to 0° C. 3.63 g (16 mmol) of5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl chloride were dissolvedin 40 mL of CH₂Cl₂ and added from a separatory funnel, drop-wise over 15min. The reaction mixture was stirred at room temperature for 3 days.The reaction mixture was transferred to a separatory funnel with CH₂Cl₂and H₂O. The water phase was thoroughly extracted with CH₂Cl₂. TheCH₂Cl₂ extract was then extracted with 0.5N HCl, dried, and evaporated.The residue was further dried in a vacuum oven to yield 5.15 g of a tansolid ofN′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazinecarboxylicacid tert-butyl ester. TLC (1:1 ethyl acetate:hexane) gave a single spotat Rf=0.43 and NMR indicated a very pure product: ¹H NMR (CDCl₃, 500MHz) δ (ppm): 7.5 (br, 1H), 7.0 (br, 1H), 6.75 (d, 2H), 4.28 (br, 4H),2.76 (m, 2H), 1.5 (s, 9H), 1.18 (t, 3H).

5.15 g (16 mmol) ofN′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazinecarboxylicacid tert-butyl ester were added to a 200 mL round bottom flask. About20 mL of trifluoroacetic acid were added and the reaction mixture wasstirred at room temperature for 24 hours. Then about 40 mL of water wereadded, followed by the slow addition of cold 10% NaOH/H₂O, withstirring, until the acid was neutralized (pH˜14). The reaction mixturewas transferred to a separatory funnel and extracted with ethyl acetateby shaking gently (caution:gas evolution). The ethyl acetate extract wasdried and evaporated to yield 5.51 g of a pale, viscous yellowsemi-solid. The material was then placed in a 50° C. vacuum oven forabout 1 hour to yield 4.62 g of5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid hydrazide. Thet-Boc cleavage is best accomplished with neat trifluoroacetic acid; useof adjunctive solvents always resulted in much lower yields. ¹H NMR(CDCl₃, 500 MHz) δ (ppm): 7.0 (br, 1H), 6.83 (m, 1H), 6.71 (m, 1H), 4.28(br s, 4H), 2.76 (m, 2H), 1.6 (br, 2H), 1.17 (t, 3H).

1.12 g (5.1 mmol) of 5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylicacid hydrazide, 1.37 g (12 mmol) of 2,2 dimethyl pentanone-3, 30 mL ofethanol, and 20 drops of glacial acetic acid were refluxed for 6 hoursto generate 5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid(1-ethyl-2,2-dimethyl-propylidene)-hydrazide, which was used in situ. Tothe cooled reaction mixture, was added 3 mL of glacial acetic acid and0.63 g (10 mmol) of NaCNBH₃. The reaction was stirred at roomtemperature for 24 hours. 25 mL of water were added and most of thealcohol was removed on a rotary evaporator. Then 10% NaOH/H₂O was addeduntil the reaction mixture was basic. The product was extracted withethyl acetate, which was then dried and evaporated to give 1.61 g ofresidue. Pure 5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acidN′-(1-ethyl-2,2-dimethyl-propyl)-hydrazide was obtained (ca. 0.77 g) bycolumn chromatography on silica gel, eluting with 25% ethylacetate/hexane. TLC: Rf=0.53, 1:1 ethyl acetate:hexane). ¹H NMR (CDCl₃,500 MHz) δ (ppm): 7.1 (br s, 1H), 6.8 (d, 1H), 6.7 (d, 1H), 4.27 (m,4H), 2.8 (m, 2H), 2.4 (m, 1H), 1.7 (m, 1H), 1.3 (m, 1H), 1.2 (t, 3H),1.15 (t, 3H), 0.97 (s, 9H).

0.214 g (0.70 mmol) of5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acidN′-(1-ethyl-2,2-dimethyl-propyl)-hydrazide, 151 mg (0.9 mmol) of 3,5dimethylbenzoyl chloride, 7 mL of 25% K₂CO₃/H₂O and 7 mL of CH₂Cl₂ wereadded to a 20 mL vial and stirred at room temperature for 24 hours. Thereaction mixture was transferred to a separatory funnel, and diluteNaHCO₃ and CH₂Cl₂ were added. The CH₂Cl₂ layer was separated and thewater layer extracted twice with CH₂Cl₂. The CH₂Cl₂ extracts were driedover MgSO₄ and evaporated to yield 0.59 g of a white residue.Purification by column chromatography and elution with 15 mL of 20%ethyl acetate/hexane yielded about 350 mg of 3,5-dimethyl-benzoic acidN′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-N-(1-ethyl-2,2-dimethyl-propyl)-hydrazide(95% pure by TLC: Rf=0.56, 1:1 ethyl acetate:hexane). ¹H NMR (CDCl₃, 500MHz) δ (ppm): 7.05 (s, 1H), 7.0 (s, 2H), 6.6 (d, 1H), 6.27 (d, 1H), 4.65(d, 1H), 4.25 (s, 4H), 2.9 (m, 1H), 2.3 (s, 6H), 2.0 (m, 1H), 1.55-1.7(m, 2H), 1.25 (m, 3H), 0.9-1.2 (3s, 9H), 0.9 (t, 3H).

The following compounds were prepared in a similar manner:

3,5-Dimethoxy-4-methyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide. TLC:Rf=0.45, 3:2 (hexane:acetone).

RG-115851, 3,5-dimethyl-benzoic acidN-(1-tert-butyl-pentyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide. ¹HNMR (CDCl₃, 500 MHz), δ (ppm): 7.7 (s, 1H), 7.22, 7.1 (2 br s, 1H), 7.08(s, 2H), 7.0 (s, 1H), 6.87 (m, 1H), 6.28 (m, 1H), 4.7 (m, 1H), 3.78 (s,3H), 2.28 (s, 6H), 1.8 (s, 3H), 1.3-1.6 (br m, 6H), 1.2, 1.1, 0.95 (3s,9H), 0.95 (m, 3H); TLC Rf=0.56 (1:1 ethyl acetate:hexane).

RG-115852, 3,5-dimethoxy-4-methyl-benzoic acidN-(1-tert-butyl-pentyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide. ¹HNMR (CDCl₃, 500 MHz), δ (ppm): 7.05 (t, 1H), 7.0 (s, 1H), 6.85 (d, 1H),6.65 (s, 2H), 6.25 (d, 1H), 4.7 (d, 1H), 3.89 (s, 3H), 3.78 (s, 6H),2.10 (s, 3H), 1.86 (s, 3H), 1.3-1.6 (br m, 6H), 1.06, 0.99 (2s, 9H),0.94 (t, 3H); TLC Rf=0.55 (1:1 ethyl acetate:hexane).

3,5-Dimethyl-benzoic acidN-(1-ethyl-2,2-dimethyl-propyl)-N′-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide.¹H NMR (CDCl₃, 500 MHz), δ (ppm): 7.05 (s, 2H), 7.0 (s, 1H), 6.6 (d,1H), 6.3 (d, 1H), 4.6 (d, 1H), 4.25 (m, 4H), 2.25 (s, 6H), 1.85 (s, 3H),1.5-1.8 (br, 2H), 1.3 (t, 3H), 1.0-1.2 (2s, 9H); TLC Rf=0.52 (1:1 ethylacetate:hexane).

3,5-Dimethoxy-4-methyl-benzoic acidN-(1-ethyl-2,2-dimethyl-propyl)-N′-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide.¹H NMR (CDCl₃, 500 MHz), δ (ppm): 6.8 (br s, 1H), 6.62 (s, 1H), 6.6 (d,1H), 6.27 (d, 1H), 4.6 (d, 1H), 4.25 (m, 4H), 3.84, 3.78 (2s, 6H), 2.1(s, 3H), 1.87 (s, 3H), 1.6 (br, 2H), 1.3 (t, 3H), 0.9-1.2 (m, 9H); TLCRf=0.45 (1:1 ethyl acetate:hexane).

3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide.¹H NMR (CDCl₃, 500 MHz), δ (ppm): 7.05 (s, 2H), 7.0 (s, 1H), 6.6 (d,1H), 6.3 (d, 1H), 4.7 (d, 1H), 4.2 (m, 4H), 2.3 (s, 6H), 1.8 (s, 3H),1.3-1.7 (br m, 4H), 1.1, 1.15 (2s, 9H), 0.95 (t, 3H).

3,5-Dimethoxy-4-methyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(5-methyl-2,3-dihydrobenzo[1,4]dioxine-6-carbonyl)-hydrazide.¹H NMR (CDCl₃, 500 MHz), δ (ppm): 6.75 (br s, 1H), 6.62 (s, 1H), 6.6 (d,1H), 6.25 (d, 1H), 4.7 (t, 1H), 4.25 (m, 4H), 3.78, 3.84 (2s, 6H), 2.85(br, 1H), 2.37 (m, 1H), 2.07 (s, 3H), 1.86 (s, 3H), 1.3-1.7 (br m, 4H),

3,5-Dimethoxy-4-methyl-benzoic acidN′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-N-(1-ethyl-2,2-dimethyl-propyl)-hydrazide.¹H NMR (CDCl₃, 500 MHz), δ (ppm): 6.8 (br s, 1 h), 6.65 (s, 1H), 6.6 (d,1H), 6.25 (d, 1H), 4.6 (d, 1H), 4.25 (2s, 4H), 3.79-3.84 (2s, 6H), 2.9(br, 1H), 2.35 (br, 1H), 2.1 (s, 3H), 1.3-1.9 (br m, 2H), 1.3 (t, 3H),1.1-1.3 (m, 9H), 0.94 (t, 3H); TLC Rf=0.48 (1:1 ethyl acetate:hexane).

3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide.¹H NMR (CDCl₃, 500 MHz), δ (ppm): 7.05 (s, 2H), 7.0 (s, 1H), 6.59 (d,1H), 6.18 (d, 1H), 4.7 (d, 1H), 4.27, 4.25 (s, 4H), 2.85 (m, 1H), 2.3(s, 6H), 2.1 (m, 1H), 1.3-1.8 (br m, 4H), 1.1, 1.15 (2s, 9H), 0.95 (t,6H); TLC Rf=0.53 (1:1 ethyl acetate:hexane).

3,5-Dimethoxy-4-methyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide.¹H NMR (CDCl₃, 500 MHz), δ (ppm): 6.75 (br s, 1H), 6.62 (s, 1H), 6.6 (d,1H), 6.2 (d, 1H), 4.7 (m, 1H), 4.25 (br m, 4H), 3.84, 3.78 (2s, 6H), 2.4(m, 1H), 1.95 (m, 1H), 2.1 (s, 3H), 1.2-1.8 (br m, 4H), 1.1-0.95 (m,9H), 0.95 (m, 6H). TLC: 0.54 (1:1 ethyl acetate:hexane); TLC Rf=0.54(1:1 ethyl acetate:hexane).

1.48 Preparation of RG-115665

Benzyl carbazate (25 g, 0.15 mol) was dissolved in 50 mL of DMF in around bottom flask. The solution was heated to 95-100° C. From twoseparate addition funnels, ethyl 2-bromoisobutyrate (58.5 g, 0.3 mol)and pyridine (29.7 g, 0.375 mol, 30 mL) were added drop-wise separatelyand simultaneously over 30-120 minutes. Heating was continued ifnecessary to propel the reaction to completion. The reaction wasmonitored by TLC (30% ethyl acetate in hexanes, I₂ visualization). Themixture was allowed to cool, and then poured onto ice water. The aqueousmixture was extracted with ethyl ether, and the solvent was removed invacuo. 2-(N′-benzyloxycarbonyl-hydrazino)-2-methyl-propionic acid ethylester was isolated, optionally after silica gel chromatography. ¹H NMR(300 MHz, CDCl₃) δ (ppm): 7.36 (br s, SH), 6.6 (br s, 1H), 5.15 (br s,2H), 4.2 (q, 2H), 1.3 (s, 6H), 1.25 (t, 3H). ¹H NMR analysis alone isinsufficient to ascertain the extent of the reaction.

2-(N′-benzyloxycarbonyl-hydrazino)-2-methyl-propionic acid ethyl ester(28 g, 0.1 mol) was dissolved in 50 mL CH₂Cl₂ and cooled on ice. Asolution of 20.7 g K₂CO₃ in 30 mL of water was added. A solution of3,5-dimethylbenzoyl chloride (17 g, 0.1 mol) in 50 mL of CH₂Cl₂ wasadded drop-wise over a period of 1 hour, maintaining the temperature at0-5° C. The mixture was stirred for 1 hour on an ice bath, and then atroom temperature overnight. TLC indicated the reaction was complete. Theaqueous layer was removed in a separatory funnel and the organic phasewas washed with water and then brine, and then dried over Na₂SO₄. Thesolvent was removed on a rotary evaporator. The residue was slurried inhexane, filtered, and then air-dried.2-[N′-Benzyloxycarbonyl-N-(3,5-dimethyl-benzoyl)-hydrazino]-2-methyl-propionicacid ethyl ester was obtained as a white solid (35 g), giving a singlespot by TLC. ¹H NMR (300 MHz, CDCl₃) δ (ppm): 7.4 (s, 1H), 7.3 (m, 3H),7.15 (m, 2H), 7.1 (s, 2H), 7.0 (s, 1H), 5.2 (d, 1H), 5.0 (d, 1H), 4.2(m, 2H), 2.25 (s, 6H), 1.78 (s, 3H), 1.64 (s, 3H), 1.28 (t, 3H).

To a 500 mL round bottom flask was added 20.62 g (0.05 mol) of2-[N′-benzyloxycarbonyl-N-(3,5-dimethyl-benzoyl)-hydrazino]-2-methyl-propionicacid methyl ester and 200 mL of dry THF. The mixture was stirred and theflask was cooled in dry ice, and then 0.87 g (0.04 mol) of LiBH₄ wasadded with stirring at room temperature. The reaction mixture wasrefrigerated and more LiBH₄ was added (1.3 g) and the reaction wasrefrigerated for 2 days. The reaction mixture was warmed to roomtemperature, and 100 mL of ether were added and the total mixture waspoured slowly into 150 mL of water in a separatory funnel. After thebubbling subsided, the mixture was agitated and then shaken gently. Theether layer was separated and the water extracted twice with 100 mL ofet2o. The total ether extract was extracted with water, washed withbrine, and evaporated to yield 20.04 g of product. The product waspurified by chromatography. The product eluted with 30-35% ethylacetate:hexane to yield 11.6 g ofN′-(3,5-dimethyl-benzoyl)-N′-(2-hydroxy-1,1-dimethyl-ethyl)-hydrazinecarboxylicacid benzyl ester, ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.3-6.9 (3s, 8H),5.1 (d, 1H), 4.9 (d, 1H), 4.1 (d, 1H), 4.1 (d, 1H), 3.6 (d, 1H), 2.25(s, 6H), 1.48 (s, 3H), 1.41 (s, 3H), as well as 4 g of unreactedstarting material.

To a round bottom flask was added 4.00 g (0.0108 mol) ofN′-(3,5-dimethyl-benzoyl)-N′-(2-hydroxy-1,1-dimethyl-ethyl)-hydrazinecarboxylicacid benzyl ester, 3.27 g (0.048 mol) of imidazole and 20 mL of DMF. Theflask was cooled in an ice bath and then 4.08 g (0.027 mol) of t-butyl,dimethylsilyl chloride was slowly added as the temperature wasmaintained below 25° C. The ice bath was removed and the reactionstirred overnight at room temperature. The reaction mixture was thenpoured into 200 mL of water and extracted three times with 100 mL ofether. The ether extract was washed with water, dried over MgSO₄, andconcentrated to yield 7.12 g of product. The product was cleaned up bycolumn chromatography. Unreacted t-butyl, dimethylsilyl chloride waseluted with hexane and 10% CH₂Cl₂/hexane. The product eluted with20-100% CH₂Cl₂/hexane to yield 5.13 g ofN′-[2-(tert-butyl-dimethyl-silanyloxy)-1,1-dimethyl-ethyl]-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylicacid benzyl ester. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.3-6.7 (m, 8H), 5.0(s, 2H), 4.1 (d, 1H), 3.4 (d, 1H), 2.15 (d, 6H), 1.54 (d, 3H), 1.25 (d,3H), 0.82 (s, 9H), 0.01 (s, 6H).

Into a 200 mL round bottom flask, was added 4.62 g (0.0095 mol) ofN′-[2-(tert-butyl-dimethyl-silanyloxy)-1,1-dimethyl-ethyl]-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylicacid benzyl ester, 100 mL of dry CH₂Cl₂, 2.5 g of Et₃N, and 1.66 g(0.0143 mol) of Et₃Si H. The reaction mixture was cooled in an ice bath,and then 100 mg of palladium acetate was added in 3 portions over 30min. The reaction was then allowed to warm to room temperature. As TLCindicated no product, only the starting material (20% ethylacetate/hexane, Rf 4.3), the reaction was warmed gently with a heat gunand then stirred at room temperature for 30 min. TLC indicated theproduct (20% ethyl acetate/hexane, Rf 0.43).

The reaction mixture was stirred with some MgSO₄ and then filtered. Thefilter cake was then washed with CH₂Cl₂. The total CH₂Cl₂ fraction wasshaken with saturated NH₄Cl, then with H₂O. The CH₂Cl₂ was dried andevaporated to yield 3.78 g of 3,5-Dimethyl-benzoic acidN-[2-(tert-butyl-dimethyl-silanyloxy)-1,1-dimethyl-ethyl]-hydrazide. Theproduct was purified by column chromatography, eluted with 5-10% ethylacetate/hexane. The product fractions were combined, concentrated, andplaced in warm (50° C.) vacuum oven to yield 2.96 g of solid3,5-Dimethyl-benzoic acidN-[2-(tert-butyl-dimethyl-silanyloxy)-1,1-dimethyl-ethyl]-hydrazide. ¹HNMR (CDCl₃, 300 MHz) δ (ppm): 7.0 (s, 2H), 6.9 (s, 1H), 3.80 (s, 2H),2.23 (s, 6H), 1.40 (s, 6H), 0.82 (s, 9H), 0.08 (s, 6H).

Into a 250 mL round bottom flask, was added 3.71 g (0.010 mol) of3,5-dimethyl-benzoic acidN-[2-(tert-butyl-dimethyl-silanyloxy)-1,1-dimethyl-ethyl]-hydrazide and50 mL of CH₂Cl₂, 2.11 g (0.106 mol) of 2-ethyl-3-methoxybenzoyl chlorideand a K₂CO₃ solution (4.15 g in 20 mL of H₂O) were added. The reactionmixture was stirred at room temperature overnight. The reaction mixturewas transferred to a separatory funnel and the aqueous layer extractedtwice with 50 mL of CH₂Cl₂. The organic phase was dried and concentratedto give 5.52 g of a syrupy product. The product was purified by columnchromatography, eluting in 10% ethyl acetate in hexane. Furtherpurification was achieved by triturating the product with heptane,placing the mixture into the freezer, and then either decanting theyellow solution or the more preferred method of rapidly filteringthrough a cold Buchner filter for 1-2 hours to obtain a white solidproduct, 2-ethyl-3-methoxy-benzoic acidN′-[2-(tert-butyl-dimethyl-silanyloxy)-1,1-dimethyl-ethyl]-N′-(3,5-dimethyl-benzoyl)-hydrazide(2.9 g). ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.0-6.8 (m, 5H), 6.2 (d, 1H),4.1 (d, 1H), 3.69 (s, 3H), 3.5 (d, 1H), 2.19 (s, 6H), 1.62 (s, 3H), 1.40(s, 3H), 0.9 (t, 3H), 0.76 (s, 9H).

2.78 g (0.00526 mol) of 2-ethyl-3-methoxy-benzoic acidN′-[2-(tert-butyl-dimethyl-silanyloxy)-1,1-dimethyl-ethyl]-N′-(3,5-dimethyl-benzoyl)-hydrazidewere dissolved in 24 mL of THF. The mixture was cooled in an ice bathand 6.0 mL (0.006 mol) of a 1 M tetrabutyl ammonium fluoride-solution inTHF were added. The reaction was stirred at room temperature for 5-6hours, and then 100 mL of Et₂O were added and the reaction mixture waspoured into ice water in a separatory funnel. The aqueous layer wasfurther extracted twice with 25 mL of Et₂O and the total ether layer wasdried and concentrated. TLC of the product showed a new product (Rf0.20) below some of the starting material. The product was purified bychromatography, eluting with 40-50% ethyl acetate/hexane to yield 1.42 gof pure 3,5-dimethyl-benzoic acidN′-[1-(2-ethyl-3-methoxy-phenyl)-vinyl]-N-(2-hydroxy-1,1-dimethyl-ethyl)-hydrazide.(67% yield). TLC: Rf=0.20. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.1-6.8 (m,5H), 6.0 (d, 1H), 4.3 (d, 1H), 3.78 (s, 3H), 3.5 (d 1H), 2.4 (d, 1H)2.29 (s, 6H), 2.2 (d, H), 1.56 (s, 3H), 1.45 (s, 3H), 1.00 (t, 3H).

1.09 g (0.0027 mol) of 3,5-dimethyl-benzoic acidN′-[1-(2-ethyl-3-methoxy-phenyl)-vinyl]-N-(2-hydroxy-1,1-dimethyl-ethyl)-hydrazidewere dissolved in 100 mL of CH₂Cl₂ and 1.80 g (0.0082 mol) of pyridiniumchlorochromate were added. The reaction mixture was refluxed for 2hours. After cooling, the total reaction mixture was poured onto asilica chromatography column to purify the product, 3,5-dimethyl-benzoicacidN-(1,1-dimethyl-2-oxo-ethyl)-N′-[1-(2-ethyl-3-methoxy-phenyl)-vinyl]-hydrazide.A white crystalline solid (1.04 g) was eluded with 30-35% ethylacetate/hexane. TLC indicated high purity (>95%), Rf=0.46 (1:1 ethylacetate:hexane). ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 9.6 (s, 1H), 7.15 (s,2H), 7.1 (m, 2H), 6.9 (d, 1H), 6.3 (d, 1H), 3.806 (s, 3H), 2.304 (s,6H), 2.2-2.4 (m, 2H), 1.576 (s, 3H), 1.429 (s, 3H), 1.01 (t, 3H).

Into a 20 mL vial, was added 55 mg of 3,5-dimethyl-benzoic acidN′-[1-(2-ethyl-3-methoxy-phenyl)-vinyl]-N-(2-hydroxy-1,1-dimethyl-ethyl)-hydrazide,2 mL of CH₂Cl₂, 200 mg of Et₃N. The reaction mixture was stirred andthen 17 mg of acetyl chloride were added. After stirring at roomtemperature for 2 hours, the reaction was warmed at 40° C. for 30 min.After cooling, more CH₂Cl₂ was added, and then transferred to separatoryfunnel and shaken with dilute K₂CO₃. The CH₂Cl₂ layer was dried withMgSO₄. TLC showed a major spot at Rf 38. The product was cleaned up bychromatography by eluting with 30% ethyl acetate in hexane. This yieldedabout 40 mg of acetic acid2-{N-(3,5-dimethyl-benzoyl)-N′-[1-(2-ethyl-3-methoxy-phenyl)-vinyl]-hydrazino}-2-methyl-propylester. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.1-6.8 (m, 6H), 6.1 (d, 1H),4.7-4.4 (q, 2H), 3.79 (s, 3H), 2.29 (s, 6H), 2.12 (s, 3H), 1.75 (s, 3H),1.49 (s, 3H), 0.98 (t, 3H).

Into a flask containing 1.5 g (0.0036 mol, 80% pure) of2-[N′-benzyloxycarbonyl-N-(3,5-dimethyl-benzoyl)-hydrazino]-2-methyl-propionicacid ethyl ester, was added 20 mL of dry CH₂Cl₂, 1.5 mL of Et₃N, and1.27 g (0.010 mol) of Et₃SiH. While stirring, small portions of a totalof 0.010 g of Pd(OAc)₂ was added and the reaction was stirred for 2hours at room temperature. To the reaction mixture was added 100 mL ofCH₂Cl₂ and some MgSO₄ to aid in the filtration/removal of the Pdproduct. The CH₂Cl₂ solution was shaken with saturated NH₄Cl, CH₂Cl₂dried, and evaporated to yield 1.61 g of2-[N-(3,5-dimethyl-benzoyl)-hydrazino]-2-methyl-propionic acid ethylester. The product was purified by chromatography, elution with 21-24%ethyl acetate in hexane. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.045 (s 2H),7.0 (s 1H), 4.4 (s, 2H), 2.324 (s 6H), 2.236 (s 3H), 1.487 (s, 6H).

To a flask containing 1.6 g (0.0057 mol) of2-[N-(3,5-Dimethyl-benzoyl)-hydrazino]-2-methyl-propionic acid ethylester in 30 mL of CH₂Cl₂ was added 3.5 g of Et₃N and 1.20 g (0.0060 mol)of 2-ethyl-3-methoxybenzoyl chloride. The reaction mixture was refluxedfor 3 hours and then evaporated to dryness. The residue was redissolvedwith 100 mL of CH₂Cl₂ and extracted twice with 50 mL of dilute aqueousK₂CO₃. The CH₂Cl₂ extract was dried and evaporated to a residue, whichwas then triturated with 6% Et₂O in hexane. A white solid product,2-[N-(3,5-dimethyl-benzoyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazino]-2-methyl-propionicacid ethyl ester, was filtered off and dried in a warm (50%) vacuumoven. TLC: product, Rf=0.40, starting material, Rf=0.35, 1:1 ethylacetate:hexane. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.70 (s 1H), 7.2-6.8(m⁻5H), 6.2 (d 1H), 4.2 (q 2H), 3.801 (s 3H), 2.4 (q 2H), 2.291 (s 6H),1.882 (s 3H), 1.557 (s 3H), 1.291 (t, 3H), 1.036 (t, 3H).

2-[N-(3,5-Dimethyl-benzoyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazino]-2-methyl-propionicacid was prepared by oxidation of 2-ethyl-3-methoxy-benzoic acidN′-(3,5-dimethyl-benzoyl)-N′-(1,1-dimethyl-2-oxo-ethyl)-hydrazide withKMnO₄. The corresponding ester could not be saponified to the acidshown, even with 40% NaO/CH₃OH, or 50% aqueous NaOH+EtOH with reflux.

50 mg (0.000126 mol) of 2-ethyl-3-methoxy-benzoic acidN′-(3,5-dimethyl-benzoyl)-N′-(1,1-dimethyl-2-oxo-ethyl)-hydrazide wasweighed into a 20 mL vial. 20 mg of hydroxylamine-HCl dissolved in 0.5mL of CH₃OH was then added. 40 mg of triethylamine was added and thereaction mixture stirred at room temperature overnight. The reactionmixture was concentrated to dryness with N₂, re-dissolved with 5 mL ofCH₂Cl₂ and 5 mL of 0.1N HCl/H₂O. The CH₂Cl₂ layer was separated. TLCshowed the product, 2-ethyl-3-methoxy-benzoic acidN′-(3,5-dimethyl-benzoyl)-N′-(2-hydroxyimino-1,1-dimethyl-ethyl)-hydrazide,had a Rf of 0.27 (1:1 ethyl acetate:hexane) and was about 85% pure. ¹HNMR (CDCl₃, 300 MHz) δ (ppm): 7.1-6.8 (m, 5H), 6.2 (d, 1H), 3.79 (s,3H), 2.29 (s, 6H), 1.76 (s, 3H), 1.65 (s, 3H), 0.98 (t, 3H).

50 mg of 2-ethyl-3-methoxy-benzoic acidN′-(3,5-dimethyl-benzoyl)-N′-(1,1-dimethyl-2-oxo-ethyl)hydrazide, 40 mgof semicarbazide, 40 mg of Et₃N and 2 mL of CH₃OH were refluxed for 2hours, concentrated to dryness, redissolved with CH₂Cl₂ and diluted with(0.5N)HCl. The CH₂Cl₂ extract was dried and evaporated to give thesemicarbazide of 2-ethyl-3-methoxy-benzoic acidN′-(3,5-dimethyl-benzoyl)-N′-(1,1-dimethyl-2-oxo-ethyl)-hydrazide. ¹HNMR (CDCl₃, 300 MHz) δ (ppm): 7.4-6.8 (m, 5H), 6.2 (d, 1H), 3.767 (s,3H), 2.203 (s, 6H), 1.717 (s, 3H), 1.474 (s, 3H), 0.913 (t, 3H).

50 mg of 2-ethyl-3-methoxy-benzoic acidN′-(3,5-dimethyl-benzoyl)-N′-(1,1-dimethyl-2-oxo-ethyl)-hydrazidealdehyde, 2 mL of CH₃OH, 0.5 mL of a 0.5% glacial acetic acid solutionwith CH₃OH and 26 mg of oxamic hydrazide, and 40 mg of Et₃N wererefluxed for 2 hours. The solvents were removed on an evaporator and theresidue redissolved with CH₂Cl₂ and water. The CH₂Cl₂ extract was driedand evaporated. TLC showed the presence of the product, the oxamiccarbazide of 2-ethyl-3-methoxy-benzoic acidN′-(3,5-dimethyl-benzoyl)-N′-(1,1-dimethyl-2-oxo-ethyl)-hydrazide, whichhad a Rf of 0.50 while the starting aldehyde had a Rf of 0.74 (in 100%ethyl acetate). ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 8.1 (s 1H), 7.1-6.8 (m,6H), 6.1 (d, 1H), 3.667 (s, 3H), 2.3 (m, 1H), 2.19 (s, 6H), 2.00 (m,1H), 1.581 (s, 3H), 1.511 (s, 3H), 0.802 (t, 3H).

50 mL of 2-ethyl-3-methoxy-benzoic acidN′-(3,5-dimethyl-benzoyl)-N′-(1,1-dimethyl-2-oxo-ethyl)-hydrazide wereadded to a 20 mL flask, with 45 g of aminoethanol in 2 mL of CH₃OH, andthen refluxed for 2 hours. After cooling, the CH₃OH was removed on theevaporator and the residue was chromatographed. The product3,5-dimethyl-benzoic acidN′-[1-(2-ethyl-3-methoxy-phenyl)-vinyl]-N-(2-hydroxymethoxyimino-1,1-dimethyl-ethyl)-hydrazidewas eluted with 40% ethyl acetate/hexane. ¹H NMR (CDCl₃, 300 MHz) δ(ppm): 7.2-6.9 (m, 5H), 4.1 (m, 2H), 3.83 (s, 3H), 3.7 (m, 2H), 2.75 (m,2H), 2, 338 (s, 6H), 1.36 (s, 3H), 1.21-1.87 (m, 6H).

To a flask containing 0.94 g (0.002 mol) of methyl etherN′-(3,5-dimethyl-benzoyl)-N′-(2-methoxy-1,1-dimethyl-ethyl)-hydrazinecarboxylicacid benzyl ester, was added 10 mL of CH₂Cl₂, 0.87 g of Et₃SiH, 0.10 gof palladium acetate, and 1 g of Et₃N. The reaction mixture was stirredat room temperature for over 10 hours. More CH₂Cl₂ (10-20 mL) was added,and the mixture was filtered to remove the palladium. The brown CH₂Cl₂solution was treated with MgSO₄ and charcoal, then filtered andevaporated. The evaporation yielded 0.87 g of a red, oily solid. TLCindicated the presence of the product; Rf=0.44 in 1:1 ethylacetate:hexane. The product was purified by chromatography, eluted with19-20% ethyl acetate/hexane to yield 401 mg (80%) of3,5-dimethyl-benzoic acid N-(2-methoxy-1,1-dimethyl-ethyl)-hydrazideproduct. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.1 (s, 2H), 7.0 (s, 1H),3.682 (s, 2H), 3.377 (s, 3H), 2.319 (s, 6H), 1.495 (s, 3H).

To a 20 mL vial containing 90 mg of 3,5-dimethyl-benzoic acidN-(2-methoxy-1,1-dimethyl-ethyl)-hydrazide 1462 (0.00036), was added 2mL of CH₂Cl₂, 145 mg (0.00072 mol) of 2-ethyl-3-methoxybenzoyl chloride,0.5 K₂CO₃ in 3 mL of H₂O. The reaction was stirred at room temperatureovernight. The reaction mixture was transferred to separatory funnelwith 10 mL of K₂CO₃ and 50 mL of CH₂Cl₂. The CH₂Cl₂ extract was driedand evaporated to dryness. The product, 2-ethyl-3-methoxy-benzoic acidN′-(3,5-dimethyl-benzoyl)-N′-(2-methoxy-1,1-dimethyl-ethyl)-hydrazide,was purified by chromatography, eluting with 25% ethyl acetate in hexaneto yield 105 mg. TLC: Rf=0.44 (1:1 ethyl acetate:hexane). ¹H NMR (CDCl₃,300 MHz) δ (ppm): 7.8 (s, 1H), 7.1-6.8 (m, 5H), 6.2 (d, 1H), 4.0 (d,1H), 3.84 (d, 1H), 3.77 (s, 3H), 3.387 (s, 3H), 2.27 (s, 6H), 2.4-2.1(m, 2H), 1.728 (s, 3H), 1.503 (s, 3H) 0.98 (t 3H).

1.49 Preparation of Compound RG-101494

N-(5-ethyl-1,4-benzodioxan-6-carbonyl)-N′-(tert-butyl)-N′-(3-chloro-5-methylbenzoyl)hydrazinecan be prepared in accordance with U.S. Pat. No. 5,530,028. Briefly, theproduct of Example 17 is treated by the method of Example 5 and then themethod of Example 8. The resulting product is treated with3-methyl-5-chlorobenzoyl chloride [(K. Knoevenagel, Chemische Berichte28: 2045 (1895); Slootmaekers, P. J., Verbeerst, R., Bull. Soc. Chm.Belg. 77: 273-285 (1968)] according to the method of Example 9.

1.50 Preparation of Compound RG-102240

N-(3-methoxy-2-ethylbenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazinecan be prepared in accordance with Example 12 of U.S. Pat. No.5,530,028.

1.51 Preparation of Compound RG-102317

N-(5-methyl-1,4-benzodioxan-6-carbonyl)-N′-(tert-butyl)-N′-(3,5-dimethylbenzoyl)hydrazinecan be prepared in accordance with Example 3 of U.S. Pat. No. 5,530,021.

1.52 Preparation of Compound RG-115092

N-(5-methyl-1,4-benzodioxan-6-carbonyl)-N′-(2-cyano-2-propyl)-N′-(3,5-dimethoxy-4-methylbenzoyl)hydrazinecan be prepared by a method directly analogous to Examples 802 and 809of U.S. Pat. No. 5,117,057 but usingN-5-methyl-1,4-benzodioxan-6-carbohydrazine (for preparation see U.S.Pat. No. 5,530,021, Example 2) and 3,5-methoxy-4-methylbenzoyl chloride.

1.53 Preparation of Compound RG-115575

3,4,5-Trifluoro-benzoic acidN-tert-butyl-N′-(5-methyl-chroman-6-carbonyl)-hydrazide can be preparedby analogy to Example 11 of U.S. Pat. No. 5,530,021, but using3,4,5-trifluorobenzoyl chloride.

1.54 Preparation of Compound RG-115637

5-Methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acidN′-tert-butyl-N′-(3,5-dimethoxy-4-methyl-benzoyl)-hydrazide can beprepared by analogy to Example 3 of U.S. Pat. No. 5,530,021, but using3,5-dimethoxy-4-methylbenzoyl chloride.

Example 2 Determination of Physical and Transport Properties

2.1 Determination of LC logP (Experimental)

1000 ppm solutions for each of a set of logP standards (compounds forwhich logP is known experimentally; see Table 3) and for each testcompound are prepared. Liquid chromatography retention times (RT) aremeasured for each substance using the conditions described below. Alinear equation is derived relating RT to logP is developed from thedata for the logP standards. The logP for the test compound iscalculated from the logP/retention time equation.

Chromatographic Conditions:

Column: MetaChem Polaris A-18 3u 50 × 3.0 mm; part no C2001-050x030Solvent Gradient: time (min.) methanol (%) water (%) 0.0 25 75 7.0 99 18.0 25 75 Temperature: (° C.): 30 Detector Type: UV or DAD (diode arraydetector): 200-220 nm2.2 Determination of C logP

ClogP can be calculated according to standard calculations known tothose of skill in the art. Exploring QSAR: Fundamentals and Applicationsin Chemistry and Biology. Corwin Hansch, Albery Leo, American ChemicalSociety, Washington, D.C., 1995

2.3 Determination of Water Solubility

Aqueous solutions are prepared as follows in triplicate: 50 μl of a10,000 ppm solution (2,000 μg of solid dissolved in 200 μl of methanol)of the substrate in methanol or DMSO is added to 1 mL of de-ionizedwater in a 2 dram or smaller vial with magnetic stirring. Stirring iscontinued overnight at ambient temperature. The slurry is taken up intoa syringe with a luer tip. The contents are passed through a new 13 mm0.2 μM Acrodisc filter (tuffryn or glass fiber) into an autosamplerbottle. For preparation of calibration standard solutions: dilutions ofthe 10,000 ppm solution were prepared at 10, 5, 1, 0.5, and 0.2 ppm. Thewater solubility of most diacylhydrazines falls within thisconcentration range. For more soluble materials, dilution of the samplesinto this range is preferable to increasing the calibration rangebecause the non-linearity of the response results in less usefulcalibration curves. However a shift in the range of calibrationstandards is required for very insoluble compounds.

Chromatography of the samples was then preformed. For mostdiacylhydrazines the following conditions were adequate for themeasurement. Other columns and gradients may be substituted asappropriate.

Column MetaChem Polaris A-18 3μ 50 × 3.0 mm; part no C2001-050x030 (orMetaChem Inertsil 5μ ODS3 50 mm × 2.1 mm) Solvent Gradient time (min.)methanol (%) water (%) 0.00 25 75 4.50 99 1 6.00 99 1 Temperature (° C.)30

Analysis of the test samples is conducted as follows: Each solubilityreplicate is analyzed in duplicate. While any suitable analysis methodis acceptable, these results were obtained by LC/MS on a MicromassPlatform II in the electrospray negative ion mode using SIM (single ionmonitoring). Standard curves are obtained from duplicate injections ofthe standards. The concentration of the substrate is determined bycalculation from the equation relating concentration and response.

2.4 Determination of Cell Permeation Coefficients

The method to determine cell permeation coefficients is known to thoseof skill in the art. MI-QSAR: Predicting Caco-2 Cell PermeationCoefficients of Organic Molecules using Membrane-Interaction QSARAnalysis. Kulkami, Amit; Han, Yi; Hopfinger, A. J.; Journal of ChemicalInformation and Computer Sciences (2002) 42: 331-342. Table 2 representsthe physical and transport properties of the compounds of the presentinvention.

TABLE 2 Physical and Transport Properties of Compounds MI- Exp. QSAR MI-LC LogP Water P(caco2) × QSAR Compound (exp) C LogP Sol. (μM)10{circumflex over ( )}6 cm/sec Log BB RG-115009 1.9 2.19 30.4 NA NARG-115613 2.8 0.82 NA NA NA RG-101523 4.35 4.46 13.4 9.3 −0.63 RG-1013824.4 4.96 NA NA NA RG-101494 4.3 4.43 NA NA NA RG-102240 4.2 4.18  9.811.4 −1.00 RG-102317 3.6 4.21 10.1 0.13  0.13 RG-103309 5.89  2.9 NA NARG-115092 3.5 2.8 NA NA NA RG-115517 3.15 2.68 36.7 0.04  0.04 RG-1155754.1 4.22 21.6 12 12.0  RG-115637 3.54 3.6 13.7 6.1 6.1 NA = not assayed

TABLE 3 Retention Times (RT) and logP for Diacylhydrazine standards RTCompound (min.) logP

  RG-102208 2.59 2.9

  RG-100864 3.29 3.2

  RG-102398 3.82 3.7

  RG-115002 3.81 3.5

  RG-102125 4.84 4.2

  RG-115372 5.52  5.02

  RG-102240 4.64 4.2

2.5 Aqueous Solubility

Equilibrium solubility was measured in pH 7.4 aqueous buffer. The bufferwas prepared by adjusting the pH of a 0.07 M solution of NaH₂PO₄ to pH7.4 with 10 N NaOH. The buffer had an ionic strength of 0.15. At least 1mg of powder was combined with 1 mL of buffer to make .gtoreq.1 mg/mLmixture. These samples were shaken for .gtoreq.2 hours and left to standovernight at room temperature. The samples were then filtered through a0.45-μm Nylon syringe filter that was first saturated with the sample.The filtrate was sampled twice, consecutively. The filtrate was assayedby HPLC against standards prepared in methanol.

TABLE 4 Solubility of Compounds Solubility (mg/mL) Compound pH 7.4RG-115280 0.0012 RG-102125 0.0006 RG-102398 ≦0.0002 RG-100150 ≧1.0RG-115595 0.026 RG-103309 ≦0.0002 RG-115555 ≧1.0 RG-115199 0.0064RG-115823 0.0003 RG-101523 0.0010 RG-102240 0.0007 RG-102317 0.0043RG-115517 0.014 RG-100021 ≧1.0

2.6 Partition Coefficients

The partition coefficient, Log (D), between water-saturated 1-octanoland pH 7.4 buffer was determined for the test compounds. The buffer wasprepared as described in section 2. A 12 μl aliquot of a 10 mM stocksolution in DMSO was introduced to a vial containing 0.60 mL of octanoland 0.60 mL of buffer at room temperature. Testosterone was also addedto a final concentration of 100 μM as an internal control. The solutionwas vortexed for 60 minutes and centrifuged at 10,000 rpm for 10minutes. The organic and aqueous layers were removed. Serial dilutionsof the organic layer were made with 50% methanol except for the initialdilution, which was made in 100% methanol. Serial dilutions of theaqueous layer were made in the pH 7.4 buffer. The diluted samples werethen assayed by LC/MS for the compound as well as for testosterone. TheLog of the ratio of peak area responses was calculated to obtain the Log(D). Typical LogD values for testosterone are from 3.0-3.3.

TABLE 5 Log (D) of Compounds Log(D) Compound Octanol/pH 7.4 RG-1152803.9 RG-102125 3.2 RG-102398 3.5 RG-100150 −2.1 RG-115595 2.1 RG-1033093.0 RG-115555 0.0 RG-115199 2.0 RG-115823 3.3 RG-101523 2.9 RG-1022403.4 RG-102317 3.6 RG-115517 2.9 RG-100021 0.8

2.7 Bi-Directional Permeability, CACO-2

Caco-2 monolayers were grown to confluence on collagen-coated,microporous, polycarbonate membranes in 12-well Costar Transwell plates.Details of the plates and their certification are shown below. Thepermeability assay buffer was Hank's Balanced Salt Solution containing10 mM HEPES and 15 mM glucose at a pH of 7.0.±0.2. The dosing solutionconcentration was 10 μM in assay buffer. At each time point, 1 and 2hours, a 200-L aliquot was taken from the receiver chamber and replacedwith fresh assay buffer. Cells were dosed on the apical side (A-to-B) orbasolateral side (B-to-A) and incubated at 37° C. with 5% CO₂ and 90%relative humidity. Each determination was performed in duplicate. Theimportant experimental parameters are outlined below. Permeabilitythrough a cell-free (blank) membrane was studied to determinenon-specific binding and free diffusion of the compound through thedevice. Lucifer yellow flux was also measured for each monolayer afterbeing subjected to the test compounds to ensure no damage was inflictedto the cell monolayers during the flux period.

All samples were assayed by LC/MS using electrospray ionization. TypicalLC/MS conditions are as follows:

Liquid Chromatography

Column: Keystone Hypersil BDS C18 30×2.0 mm i.d., 3 μm, with guardcolumn

M.P. Buffer: Ammonium Formate Buffer, pH 3.5

Aqueous Reservoir (A): 90% water, 10% bufferOrganic Reservoir (B): 90% acetonitrile, 10% buffer

Flow Rate: 300 μL/min.

Gradient Program Grad. (typically):

Time Grad. Curve % A % B TE3 TE4 −0.1 0 100 0 close 1.2 1 60 40 close3.0 1 0 100 3.1 0 100 0 4 0 100 0 closeTotal run time: 4.5 minAutosampler: 10 μL injection volumeAutosampler wash: water/acetonitrile/2-propanol::1/1/1; with 0.2% formicacid

Mass Spectrometer (Typical Operating Conditions) Interface: Electrospray(“Turbo Ionspray”) Mode: Single Ion Monitoring

Gases: Neb Gas=8, Curtain Gas=10, Turbo Ionspray Gas=8000 mL/min. TEM:350° C. Voltages: IS 4500, OR 25, RNG 200, Q0-10, IQ1-12, ST-15, RQ0-12,DF-200, CEM (per age)Method: 4.5 minute duration.

The apparent permeability, Papp, and percent recovery were calculated asfollows:

Papp=(dC _(r) /dt)×V _(r)/(A×C ₀)  (1)

Percent Recovery=100×((V_(r) ×C _(r) ^(final))+(V _(d) ×C _(d)^(final)))/(V _(d) ×C ₀)  (2)

where,

-   -   dC_(r)/dt is the cumulative concentration in the receiver        compartment versus time in M s⁻1.    -   V_(r) is the volume of the receiver compartment in cm³.    -   V_(d) is the volume of the donor compartment in cm³.    -   A is the area of the cell monolayer (1.13 cm² for 12-well        Transwell).    -   C₀ is the concentration of the dosing solution in M.    -   C_(r) ^(final) is the cumulative receiver concentration in M at        the end of the incubation period.    -   C_(d) ^(final) is the concentration of the donor in M at the end        of the incubation period.

Plates: TW12 TW12 Seed Date: Jun. 11, 2002 (KW) Jun. 18, 2002 (PSK)Passage: 62 61 Age (days): 27 22

Certification Acceptance Criteria TEER Value (Ω · cm²): 506 504 450-650Ω · cm² Lucifer Yellow, 0.14 0.12 <0.4 × 10⁻⁶ cm/s Papp × 10⁻⁶ cm/s:Atenolol, Papp × 10⁻⁶ cm/s: 0.20 0.18 <0.5 × 10⁻⁶ cm/s Propranolol, 2019 15-25 × 10⁻⁶ cm/s Papp × 10⁻⁶ cm/s: Digoxin, Papp × 10⁻⁶ cm/s: 1.71.8 none Digoxin, Papp × 10⁻⁶ cm/s: 12 16 none

Experimental Parameters Dosing Concentration: 10 μM Replicates: 2

Direction: apical-to-basolateral, basolateral-to-apicalTime Points: 1 and 2 hours

TABLE 6 Recovery and Permeability (10⁻⁶ cm/s) of Compounds Papp^(B-A)Percent Recovery^((C)) Papp^((D)) Papp, A-to-B Papp, B-to-A Papp^(A-B)Absorption Significant Compound Blank A-to-B B-to-A Blank Rep. 1 Rep. 2Avg Rep. 1 Rep. 2 Avg Ratio^((B)) Potential^((A)) Efflux^((B)) RG-11528041 46 99 1.88 1.24 1.30 1.27 1.50 1.48 1.49 1.2 High No RG-102125 62 8463 21.9 25.7 26.3 26.0 20.7 19.7 20.2 0.8 High No RG-102398 94 85 9034.7 27.0 26.8 26.9 25.9 28.2 27.1 1.0 High No RG-100150 117 102 10736.9 0.19 0.18 0.18 0.33 0.34 0.33 1.8 Low No RG-115595 103 95 104 33.219.6 18.9 19.2 29.8 31.7 30.7 1.6 High No RG-103309 70 74 75 23.6 25.223.8 24.5 32.5 32.5 32.5 1.3 High No RG-115555 110 98 106 35.8 0.18 0.180.18 1.55 1.67 1.61 8.9 Low Yes RG-115199 82 85 87 23.0 31.1 30.2 30.631.0 30.9 30.9 1.0 High No RG-115823 77 64 63 24.2 17.4 17.8 17.6 18.815.4 17.1 1.0 High No RG-101523 95 95 88 29.4 24.2 26.6 25.4 23.6 25.424.5 1.0 High No RG-102240 78 94 87 28.4 32.3 31.4 31.8 23.3 23.7 23.50.7 High No RG-102317 87 91 86 27.9 28.6 27.3 28.0 21.0 21.2 21.1 0.8High No RG-115517 96 91 95 31.6 28.4 29.3 28.8 25.2 27.6 26.4 0.9 HighNo RG-100021 113 90 94 43.0 0.21 0.22 0.22 0.91 0.95 0.93 4.3 Low No^((A))Absorption Potential Classification: Papp(A-to-B) ≧ 1.0 × 10⁻⁶cm/s High Papp(A-to-B) > 0.5 × 10⁻⁶ cm/s, Papp < 1.0 × 10⁻⁶ cm/s MediumPapp(A-to-B) < 0.5 × 10⁻⁶ cm/s Low ^((B))Efflux considered significantif: Papp (B-to-A) ≧ 1.0 × 10⁻⁶ cm/s and Ratio Papp(B-to-A)/Papp(A-to-B)≧ 3.0 ^((C))Low recoveries caused by non-specific binding, etc. canaffect the measured permeability ^((D))A low rate of diffusion (<20 ×10⁻⁶ cm/s) through the cell-free membrane indicates a lack of freediffusion, which may affect the measured permeability.

Example 3 Biological Testing of Compounds

The ligands of the present invention are useful in various applicationsincluding gene therapy, expression of proteins of interest in hostcells, production of transgenic organisms, and cell-based assays.

27-63 Assay Gene Expression Cassette

GAL4 DBD (1-147)-C/EcR(DEF)/VP16AD-βRXREF-LmUSPEF: The wild-type D, E,and F domains from spruce budworm Cloristoneura fumiferana EcR(“CfEcR-DEF”; SEQ ID NO: 1) were fused to a GAL4 DNA binding domain(“Gal4 DBD1-147”; SEQ ID NO: 2) and placed under the control of aphosphoglycerate kinase promoter (“PGK”; SEQ ID NO: 3). Helices 1through 8 of the EF domains from Homo sapiens RXRβ (“HsRXRβ-EF”;nucleotides 1-465 of SEQ ID NO: 4) and helices 9 through 12 of the EFdomains of Locusta migratoria Ultraspiracle Protein (“LmUSP-EF”;nucleotides 403-630 of SEQ ID NO: 5) were fused to the transactivationdomain from VP16 (“VP16AD”; SEQ ID NO: 6) and placed under the controlof an elongation factor-1α: promoter (“EF-1α”; SEQ ID NO: 7). Fiveconsensus GAL4 response element binding sites (“5XGAL4RE”; comprising 5copies of a GAL4RE comprising SEQ ID NO: 8) were fused to a syntheticTATA minimal promoter (SEQ ID NO: 9) and placed upstream of theluciferase reporter gene (SEQ ID NO: 10).

Stable Cell Line

CHO cells were transiently transfected with transcription cassettes forGAL4 DBD (1-147) CfEcR(DEF) and for VP16AD βRXREF-LmUSPEF controlled byubiquitously active cellular promoters (PGK and EF-1α, respectively) ona single plasmid. Stably transfected cells were selected by Zeocinresistance. Individually isolated CHO cell clones were transientlytransfected with a GAL4 RE-luciferase reporter (pFR Luc). 27-63 clonewas selected using Hygromycin.

Treatment with Ligand

Cells were trypsinized and diluted to a concentration of 2.5×10⁴ cellsmL. 100 μL of cell suspension was placed in each well of a 96 well plateand incubated at 37° C. under 5% CO₂ for 24 h. Ligand stock solutionswere prepared in DMSO and diluted 300 fold for all treatments. Doseresponse testing consisted of 8 concentrations ranging from 33 μM to0.01 μM.

Reporter Gene Assay

Luciferase reporter gene expression was measured 48 h after celltreatment using Bright-Glo™ Luciferase Assay System from Promega(E2650). Luminescence was detected at room temperature using a Dynex MLXmicrotiter plate luminometer.

Z3 Assay Stable Cell Line

Dr. F. Gage provided a population of stably transformed cells containingCVBE and 6XEcRE as described in Suhr, S. T., Gil, E. B., Senut M. C.,Gage, F. H. (1998) Proc. Natl. Acad. Sci. USA 95, 7999-804. Human 293kidney cells, also referred to as HEK-293 cells, were sequentiallyinfected with retroviral vectors encoding first the switch constructCVBE, and subsequently the reporter construct 6XEcRE Lac Z. The switchconstruct contained the coding sequence for amino acids 26-546 fromBombyx mori EcR (BE) (Iatrou) inserted in frame and downstream of theVP16 transactivation domain (VBE). A synthetic ATG start codon wasplaced under the control of cytomegalovirus (CVBE) immediate earlypromoter and flanked by long terminal repeats (LTR). The reporterconstruct contained six copies of the ecdysone response element (EcRE)binding site placed upstream of LacZ and flanked on both sides with LTRsequences (6XEcRE).

Dilution cloning was used to isolate individual clones. Clones wereselected using 450 ug/mL G418 and 100 ng/mL puromycin. Individual cloneswere evaluated based on their response in the presence and absence oftest ligands. Clone Z3 was selected for screening and SAR purposes.

Human 293 kidney cells stably transformed with CVBE and 6XEcRE LacZ weremaintained in Minimum Essential Medium (Mediates, 10-010-CV) containing10% FBS (Life Technologies, 26140-087), 450 gum G418 (Mediates,30-234-CR), and 100 gnome promising (Sigma, P-7255), at 37° C. in anatmosphere containing 5% CO₂ and were subculture when they reached 75%confluence.

Treatment with Ligand

Z3 cells were seeded into 96-well tissue culture plates at aconcentration of 2.5×10³ cells per well and incubated at 37° C. in 5%CO₂ for twenty-four hours. Stock solutions of ligands were prepared inDMSO. Ligand stock solutions were diluted 100 fold in media and 50 μL ofthis diluted ligand solution (33 μM) was added to cells. The finalconcentration of DMSO was maintained at 0.03% in both controls andtreatments.

Reporter Gene Assays Reporter gene expression was evaluated 48 hoursafter treatment of cells, β-galactosidase activity was measured usingGal Screen™ bioluminescent reporter gene assay system from Tropix(GSY1000). Fold induction activities were calculated by dividingrelative light units (“RLU”) in ligand treated cells with RLU in DMSOtreated cells. Luminescence was detected at room temperature using aDynex MLX microtiter plate luminometer.

A schematic of switch construct CVBE, and the reporter construct 6XEcRELac Z is shown in FIG. 1. Flanking both constructs are long terminalrepeats, G418 and puromycin are selectable markers, CMV is thecytomegalovirus promoter, VBE is coding sequence for amino acids 26-546from Bombyx mori EcR inserted downstream of the VP16 transactivationdomain, 6XEcRE is six copies of the ecdysone response element, lacZencodes for the reporter enzyme β-galactosidase.

13B3 Assay Gene Expression Cassette

GAL4 DBD-CfEcR(DEF)/VP16AD-MmRXRE: The wild-type D, E, and F domainsfrom spruce budworm Choristoneura fumiferana EcR (“CfEcR-DEF”; SEQ IDNO: 1) were fused to a GAL4 DNA binding domain (“Gal4 DBD1-147”; SEQ IDNO: 2) and placed under the control of the SV40e promoter of pM vector(PT3119-5, Clontech, Palo Alto, Calif.). The D and E domains from MusMusculus RXR (“MmRXR-DE”; SEQ ID NO: 11) were fused to thetransactivation domain from VP16 (“VP16AD”; SEQ ID NO: 6) and placedunder the control of the SV40e promoter of the pVP16 vector (PT3127-5,Clontech, Palo Alto, Calif.).

Stable Cell Line

CHO cells were transiently transfected with transcription cassettes forGAL4 DBD-CJEcR(DEF) and for VP16AD-MmXRXRE controlled by SV40epromoters. Stably transfected cells were selected using Hygromycin.Individually isolated CHO cell clones were transiently transfected witha GAL4 RE-luciferase reporter (pFR-Luc, Stratagene, La Jolla, Calif.).The 13B3 clone was selected using Zeocin.

Treatment with Ligand

Cells were trypsinized and diluted to a concentration of 2.5×10⁴ cellsmL. 100 μL of cell suspension was placed in each well of a 96 well plateand incubated at 37° C. under 5% CO₂ for 24 h. Ligand stock solutionswere prepared in DMSO and diluted 300 fold for all treatments. Doseresponse testing consisted of 8 concentrations ranging from 33 μM to0.01 μM.

Reporter Gene Assay

Luciferase reporter gene expression was measured 48 h after celltreatment using Bright-Glo™ Luciferase Assay System from Promega(E2650). Luminescence was detected at room temperature using a Dynex MLXmicrotiter plate luminometer.

The results of the assays are shown in Tables 7 and 8. Each assay wasconducted in two separate wells, and the two values were averaged. Foldinductions were calculated by dividing relative light units (“RLU”) inligand treated cells with RLU in DMSO treated cells. EC₅₀s werecalculated from dose response data using a three-parameter logisticmodel. Relative Max FI was determined as the maximum fold induction ofthe tested ligand (an embodiment of the invention) observed at anyconcentration relative to the maximum fold induction of GS-™-E ligand(3,5-dimethyl-benzoic acidN-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide) observed at anyconcentration.

TABLE 7 Biological Assay Results for Compounds: Fold Induction 13B3Assay 13B3 Assay 27-63 Assay 27-63 Assay Z3 Assay Z3 Assay Compound 3.3uM 33 uM 3.3 uM 33 uM 3.3 uM 33 uM RG-100021 0 1 RG-100127 365 49 1239245 RG-100150 1 1 RG-100216 8037 53 1312 398 RG-100342 611 2 1287 427RG-100360 1111 745 1627 1353 870 891 RG-100394 178 1339 11 RG-100425 7471156 1099 1128 592 RG-100448 2 66 847 423 RG-100492 1002 4 1378 143RG-100524 1211 991 146 RG-100568 2 4122 615 2286 276 1193 RG-100569 1710329 884 841 754 457 RG-100574 0 151 21 389 RG-100603 570 453 RG-10062013982 710 428 RG-100667 1191 1480 238 RG-100690 1094 500 1136 1047 779475 RG-100691 643 1378 1209 843 565 RG-100694 3385 2078 1057 1004 12941288 RG-100698 434 1089 591 RG-100699 2296 398 1413 415 RG-100725 5811096 1050 511 RG-100749 0 288 874 145 RG-100763 2442 107 1609 151RG-100764 3814 915 369 RG-100766 391 931 1993 921 474 RG-100767 4504 6822097 2171 825 371 RG-100768 709 1738 1852 1246 425 RG-100769 1014 3861595 1556 1096 542 RG-100778 2344 2159 2312 365 RG-100779 2979 947 304RG-100801 16 1341 1244 153 RG-100812 202 1587 399 RG-100814 423 11512441 2165 410 1279 RG-100848 3391 578 1181 1151 885 26 RG-100864 318 1871 1184 63 840 RG-100875 23 1143 34 1592 1006 1081 RG-100901 882 1527 185RG-100915 133 525 532 1292 RG-100929 359 1080 988 490 RG-101013 1084 406927 988 1039 846 RG-101016 4693 308 1347 394 RG-101036 4582 908 17211459 767 404 RG-101036 4582 908 1721 1459 767 404 RG-101048 701 19 1453283 RG-101057 33 842 30 1008 632 930 RG-101062 319 1127 391 RG-1010881400 6 1743 302 RG-101171 8453 395 1357 268 RG-101178 699 1200 431RG-101202 5 7 195 1 RG-101218 1983 1097 1223 886 398 RG-101248 415 1461033 375 RG-101312 1874 600 292 RG-101316 1280 1045 350 RG-101340 675879 1624 1549 866 432 RG-101353 1347 718 3583 3835 530 439 RG-10137625656 7393 1293 415 RG-101382 13058 1012 2704 2568 725 426 RG-101398 1591138 403 RG-101408 326 2445 1886 780 398 RG-101494 755 613 717 294 832384 RG-101509 1144 1303 328 RG-101511 1912 744 1421 1207 947 487RG-101523 831 718 2427 2745 704 596 RG-101528 259 3027 748 1515RG-101531 274 1130 428 RG-101542 439 1151 324 RG-101585 1015 154 1495330 RG-101659 1556 1185 2283 1896 954 876 RG-101664 1 0 6 1 RG-1016702297 738 1190 929 RG-101691 5245 630 2249 2073 654 1089 RG-101692 4180170 1798 591 435 860 RG-101734 918 442 987 951 RG-101759 623 137 796 158RG-101774 987 526 1807 1359 631 429 RG-101862 3279 293 717 250 RG-1018633187 207 1705 636 832 374 RG-101864 5959 349 1807 1464 796 494 RG-1018872462 542 1142 1107 1004 334 RG-101889 378 1085 245 RG-102021 4081 2951417 RG-102125 762 315 1164 1043 359 473 RG-102125 762 315 1164 1043 359473 RG-102317 2425 814 2551 2504 416 1501 RG-102398 8125 795 2875 3181535 1972 RG-102408 25 249 RG-102592 194 909 7 1265 746 574 RG-103309 92495 2537 1201 1155 591 RG-103361 504 2262 171 244 RG-103451 576 661 33263865 67 118 RG-104074 544 5378 189 200 RG-115006 4180 3146 415 1071RG-115009 19 2547 35 472 RG-115025 8 1700 2288 2243 269 489 RG-115033386 12 2 1256 24 388 RG-115038 4119 4970 600 321 RG-115043 835 547 4661588 RG-115046 1076 18 1069 754 RG-115050 2027 894 424 1446 RG-1150551356 1350 4499 2725 573 617 RG-115064 2 415 1 3 RG-115065 880 859 1095878 RG-115068 2828 1500 54 802 RG-115077 932 199 294 RG-115085 236 11431149 627 RG-115086 433 RG-115088 542 1048 561 228 RG-115092 2322 24092869 3106 351 302 RG-115095 RG-115102 1425 109 1154 971 88 865 RG-11510668 RG-115112 618 RG-115116 2 276 RG-115118 979 769 1063 914 90 1160RG-115128 110 RG-115130 987 511 4436 4096 412 930 RG-115143 1 2032 591736 111 1045 RG-115162 755 320 1814 1464 334 772 RG-115167 73 RG-115169405 443 RG-115171 RG-115191 3 386 RG-115199 349 RG-115199 349 RG-1152077260 7959 1332 1279 354 508 RG-115220 5 1143 298 RG-115223 8 3437 2731935 323 1299 RG-115229 599 709 2829 1423 1032 RG-115244 2404 573 22031847 283 816 RG-115253 471 848 910 832 RG-115256 647 RG-115257 820 3541130 1320 297 691 RG-115258 144 3745 1973 2212 382 1120 RG-115259 35132981 91 RG-115260 13 526 RG-115261 31 RG-115269 112 RG-115278 1950 10501250 906 158 1225 RG-115280 0 9 422 376 RG-115280 0 9 422 376 RG-1152971364 304 1544 946 443 604 RG-115302 521 648 940 815 404 RG-115306 5RG-115310 2 3044 199 960 RG-115311 RG-115327 3785 279 325 RG-115329 5644259 430 RG-115330 1995 3577 633 432 RG-115337 631 1010 1080 1053 682RG-115350 450 499 RG-115352 7 RG-115378 1778 2424 1493 1407 488 1464RG-115384 2753 2277 1713 1282 337 RG-115407 2476 2612 1611 1515 391 879RG-115416 3618 2737 2412 1867 164 1116 RG-115422 204 RG-115429 18 1843 31724 RG-115441 118 RG-115443 RG-115480 RG-115496 874 RG-115499 1182 7312092 1536 173 641 RG-115508 310 191 1195 969 199 680 RG-115514 4009 5151616 1427 383 658 RG-115515 1996 1306 420 RG-115517 8397 11953 408RG-115517 8397 11953 408 RG-115518 1644 926 1640 1126 232 803 RG-115532908 738 530 RG-115534 211 168 249 RG-115536 483 488 RG-115539 20 5921076 534 339 RG-115550 1 0 21 RG-115551 290 1150 1068 470 RG-115555 1RG-115557 426 RG-115567 RG-115575 3085 865 282 785 RG-115580 298 11041031 615 RG-115592 RG-115595 6 1381 44 1067 RG-115595 6 1381 44 1067RG-115609 558 4217 90 383 RG-115611 745 RG-115613 180 1726 991 1469RG-115625 3 715 265 1559 RG-115627 1291 10058 378 362 RG-115637 1109 6364159 2741 211 RG-115647 2169 98 817 815 96 510 RG-115648 9 RG-1156641363 333 1928 1768 209 620 RG-115674 442 319 RG-115683 RG-115684 151 498RG-115689 13 RG-115690 930 571 RG-115716 3 0 66 RG-115717 0 0 9RG-115718 0 2 0 RG-115719 0 1 271 RG-115721 0 0 2 RG-115722 0 0 1RG-115723 0 0 17 RG-115819 1970 2371 1433 722 RG-115820 2861 1971 1413701 RG-115823 2093 1025 1440 1050 RG-115824 2675 948 895 737 RG-1158292605 45 1441 319 RG-115830 2287 353 1604 983 RG-115831 2063 1435 1481544 RG-115832 2063 1435 1564 621 RG-115834 1900 1837 RG-115835 3 1895RG-115836 1822 823 RG-115837 1474 1156 RG-115840 1612 263 RG-115841 1407437 RG-115842 1269 447 RG-115846 1643 645 RG-115847 2729 848 RG-1158481346 1156 RG-115849 1 231 RG-115850 1 23 RG-115856 1760 RG-115857 328RG-115858 182 RG-115859 1056 RG-115861 8 593 RG-115862 1 243 RG-115863 1804 RG-115864 1255 RG-115865 76 RG-115866 3 RG-115867 654 RG-115003 0301 1276 RG-115044 0 1 1 2 1 7 RG-115079 3 0 1 0 RG-115091 1 61RG-115117 1 221 7 RG-115160 3 3 3 157 RG-115172 1 0 1 216 RG-115225 3 83 571 RG-115358 1 584 33 769 RG-115371 1 1092 249 1774 RG-115408 1 0 1 3RG-115490 2 844 123 814 RG-115497 1 0 1 20 RG-115511 1 98 1 1155 10 570RG-115597 0 4667 2 2065 942 RG-115653 6 0 1 832 666 RG-115665 1 0 2 0 674 RG-115783 3 2765 1362

TABLE 8 Biological Assay Results for Compounds: EC50 Relative Max FI13B3 EC50 13B3 assay Rel 27-63 assay 27-63 assay Z3 assay Z3 assayCompound (μM) Max FI EC50 (μM) Rel Max FI EC50 (μM) Rel Max FI RG-1001276.704 0.689 4.677 RG-100216 6.972 0.685 1.778 RG-100342 17.694 0.67410.233 RG-100360 0.588 0.825 0.615 0.873 0.257 1.073 RG-100394 1.223RG-100425 0.370 0.671 0.099 1.180 RG-100448 5.288 0.609 2.818 RG-10049211.757 0.916 8.128 RG-100524 0.263 1.002 RG-100568 44.284 1.035 4.1321.197 3.311 0.855 RG-100569 0.185 0.773 0.287 0.720 0.098 0.835RG-100574 4.000 0.371 1.122 RG-100620 2.399 0.869 RG-100667 0.347 0.890RG-100690 0.268 0.506 0.200 0.727 0.240 1.008 RG-100691 0.330 0.8630.257 0.945 RG-100694 0.405 0.632 0.400 0.767 0.389 1.157 RG-1006981.000 1.025 RG-100699 3.963 0.939 2.089 RG-100725 0.330 0.726 RG-1007492.692 0.951 RG-100763 5.301 0.856 2.138 RG-100764 2.344 0.900 RG-1007663.419 0.944 0.174 1.194 RG-100767 1.056 0.920 0.334 1.218 0.251 0.996RG-100768 0.333 1.039 0.214 1.129 RG-100769 0.337 0.685 0.196 0.7350.219 1.109 RG-100778 0.678 1.342 0.513 1.000 RG-100779 0.178 0.707RG-100801 2.076 0.936 0.891 RG-100812 0.912 0.954 RG-100814 37.888 0.8431.500 1.157 1.148 RG-100848 1.645 0.666 0.322 0.765 0.240 0.996RG-100864 1.155 17.269 0.716 2.239 RG-100875 4.019 0.853 8.920 1.0042.042 0.818 RG-100901 0.316 0.918 RG-100915 4.449 0.552 1.000 0.912RG-100929 1.047 0.770 0.631 RG-101013 2.256 0.539 2.140 0.606 1.4790.720 RG-101016 4.201 0.703 1.995 RG-101036 0.223 0.545 0.177 0.7240.054 1.049 RG-101036 0.223 0.545 0.177 0.724 0.054 1.049 RG-1010488.415 0.708 19.055 RG-101057 4.970 0.569 6.617 0.648 4.467 0.697RG-101062 2.570 0.992 RG-101088 9.947 1.159 4.074 RG-101171 4.019 0.8773.236 RG-101178 1.318 0.887 RG-101202 0.369 0.007 1.738 RG-101218 0.1280.645 0.316 0.998 RG-101248 5.036 0.790 1.445 RG-101312 5.623 0.955RG-101316 1.585 0.896 RG-101340 0.106 0.364 0.240 0.964 0.234 1.126RG-101353 0.675 1.163 0.266 1.168 0.107 1.078 RG-101376 1.361 0.6540.537 0.777 RG-101382 0.588 0.915 0.306 1.188 0.066 0.951 RG-1013980.282 0.712 RG-101408 0.336 1.185 0.148 1.118 RG-101494 0.148 0.8220.036 0.916 0.079 1.093 RG-101509 1.175 0.882 RG-101511 0.794 0.8110.714 0.762 0.468 1.122 RG-101523 2.374 0.772 0.165 0.943 0.242RG-101528 29.303 1.111 1.047 0.923 RG-101531 0.085 RG-101542 0.324 0.793RG-101585 5.057 0.893 4.898 RG-101659 0.562 0.867 0.347 1.009 0.214RG-101664 >50 0.002 21.081 RG-101670 0.646 0.874 0.468 0.853 RG-1016910.406 0.918 0.250 0.969 0.174 1.068 RG-101692 0.728 1.004 0.788 1.0080.309 0.975 RG-101734 0.145 0.988 RG-101759 0.877 0.336 0.331 0.639RG-101774 0.327 0.657 0.359 0.795 0.155 0.943 RG-101862 1.050 0.8610.269 0.866 RG-101863 0.728 0.837 1.003 1.043 0.257 0.953 RG-1018640.288 0.460 0.343 0.750 0.245 1.075 RG-101887 0.861 0.579 0.351 0.6750.257 0.930 RG-101889 3.300 RG-102021 4.045 1.136 0.513 0.920 RG-1021250.479 0.190 0.721 0.174 RG-102125 0.479 0.190 0.721 0.174 RG-102240 0.51 0.288 1 0.286 1 RG-102317 0.345 0.102 0.948 RG-102398 0.627 0.8380.325 1.017 0.095 1.027 RG-102408 0.091 0.662 0.054 1.114 RG-1025924.922 0.684 7.770 0.798 3.890 0.699 RG-103309 0.146 1.486 3.024 0.9750.078 0.950 RG-103361 28.000 0.758 0.526 0.938 RG-103451 0.208 0.7420.314 0.884 0.083 1.054 RG-104074 0.865 0.812 0.307 1.122 RG-1150063.496 0.890 0.275 1.031 RG-115009 18.908 0.917 3.307 0.916 RG-11502539.254 0.186 2.449 1.523 0.347 0.385 RG-115033 1.000 0.643 18.101 0.86816.218 0.575 RG-115038 1.743 1.172 0.550 1.064 RG-115043 2.669 0.8520.178 0.943 RG-115046 5.657 0.739 3.548 RG-115050 0.762 0.724 0.2820.808 RG-115055 0.303 0.894 0.318 1.341 0.043 0.936 RG-115064 >500.000 >50 0.031 RG-115065 3.000 0.746 1.479 RG-115068 0.926 0.722 21.0000.804 RG-115077 0.847 1.509 0.389 0.911 RG-115085 0.807 0.683 0.195RG-115086 0.692 0.433 RG-115088 1.827 0.709 0.501 0.958 RG-115092 0.1841.176 0.213 1.092 0.037 0.917 RG-115102 0.930 0.546 0.491 0.667 0.1390.858 RG-115106 2.570 0.457 RG-115112 0.309 0.618 RG-115116 >50 1.1723.311 0.960 RG-115118 0.689 0.530 0.394 0.612 0.234 0.969 RG-1151280.245 0.538 RG-115130 0.512 0.093 1.375 0.036 RG-115143 >50 0.651 7.7771.112 7.413 0.844 RG-115162 0.811 0.630 0.424 0.653 0.069 0.988RG-115167 0.240 0.533 RG-115169 0.497 0.618 0.347 0.956 RG-115191 >501.513 2.399 0.934 RG-115199 3.467 0.734 RG-115207 0.618 0.707 0.5930.704 0.257 0.923 RG-115220 >50 0.125 0.871 0.591 RG-115223 >50 1.3464.685 1.167 1.905 0.925 RG-115229 2.250 0.418 1.153 0.735 0.427RG-115244 0.369 0.663 0.169 0.785 0.085 0.959 RG-115253 0.107 0.7420.269 RG-115256 0.692 0.527 RG-115257 0.886 0.742 0.698 0.663 0.2040.885 RG-115258 27.111 1.334 1.879 1.279 0.646 0.882 RG-115259 2.7040.702 0.427 0.617 RG-115261 29.512 0.498 RG-115269 0.437 0.552 RG-1152780.630 0.823 0.885 0.720 0.257 0.893 RG-115280 11.000 0.254 RG-11528011.000 0.254 RG-115297 1.361 0.737 0.330 0.986 0.066 0.872 RG-1153022.000 0.624 1.044 0.521 0.275 RG-115306 3.467 0.424 RG-115310 >50 0.6762.630 0.781 RG-115327 2.754 0.662 RG-115329 2.570 0.892 RG-115330 5.6900.711 0.186 0.860 RG-115337 0.621 0.927 0.363 0.822 0.095 RG-1153500.795 0.711 0.309 0.935 RG-115352 45.709 0.265 RG-115378 1.762 0.6550.758 0.863 0.324 0.947 RG-115384 0.320 0.426 0.113 0.645 0.166 0.904RG-115407 7.000 1.048 1.056 0.932 0.347 0.906 RG-115416 0.938 12.1040.637 0.721 0.091 1.869 RG-115422 0.398 0.769 RG-115429 8.676 1.0659.574 0.880 RG-115441 0.126 0.759 RG-115496 1.175 0.878 RG-115499 0.3280.489 0.336 0.705 0.170 0.954 RG-115508 0.849 0.805 1.033 0.719 0.5370.787 RG-115514 0.541 0.550 0.170 0.720 0.056 0.970 RG-115515 0.6170.975 RG-115517 0.355 0.675 0.089 0.923 RG-115517 0.355 0.675 0.0890.923 RG-115518 1.253 0.648 1.053 0.866 0.257 0.834 RG-115532 0.5180.835 0.129 0.933 RG-115534 2.754 0.463 RG-115536 0.781 0.734 0.1260.950 RG-115539 5.000 0.955 1.177 0.595 0.151 RG-115551 0.852 0.6840.398 RG-115555 >50 0.006 RG-115557 1.698 0.566 RG-115575 0.271 0.6410.102 0.886 RG-115580 0.375 0.760 0.182 RG-115595 5.623 0.686 RG-1155955.623 0.686 RG-115609 13.782 0.883 1.386 0.625 RG-115611 1.585 0.703RG-115613 5.937 0.734 0.589 0.964 RG-115625 25.322 0.931 0.389 0.950RG-115627 0.921 0.854 0.813 0.840 RG-115637 0.088 1.002 0.117 1.3050.018 0.947 RG-115647 0.832 0.662 0.372 0.505 0.145 0.923 RG-11564821.380 0.182 RG-115664 0.323 0.497 0.359 0.635 0.126 0.846 RG-1156741.723 0.875 0.229 0.995 RG-115689 100.000 0.411 RG-115690 0.632 1.1810.209 0.998 RG-115819 0.025 1.045 0.042 0.915 RG-115820 0.193 1.2700.203 0.887 RG-115823 0.011 1.069 0.020 0.933 RG-115824 0.036 1.2180.046 0.806 RG-115829 0.035 1.264 0.058 0.937 RG-115830 0.036 1.0490.045 1.020 RG-115831 0.096 1.366 0.102 0.937 RG-115832 0.035 1.0750.037 1.002 RG-115834 1.170 0.733 RG-115835 20.000 0.731 RG-115836 0.0980.808 RG-115837 1.114 0.569 RG-115840 0.110 0.776 RG-115841 2.015 0.610RG-115842 1.196 0.524 RG-115846 0.095 0.846 RG-115847 1.100 1.120RG-115848 1.291 0.494 RG-115849 >33 0.007 RG-115850 >33 0.001 RG-1158510.02 1 RG-115852 0.07 1 RG-115856 0.003 0.879 RG-115857 0.013 1.140RG-115858 0.005 0.915 RG-115859 0.007 1.218 RG-115861 4.308 0.215RG-115862 >33 0.048 RG-115863 >33 0.154 RG-115864 0.004 0.979 RG-1158650.010 1.246 RG-115044 >50 0.87 >33 0.00 >50 1.08 RG-115079 0.02 0.20 >500.02 RG-115117 >50 0 3.93 0.36 RG-115160 >50 0 >50 0.21 RG-115172 >500 >50 0.32 RG-115225 0.58 0.08 >50 0.32 RG-115358 >50 0.20 9.45 0.48RG-115371 >50 1.44 3.02 0.76 RG-115490 >50 1.01 6.35 0.44 RG-115497 >500 >50 0.07 RG-115511 >50 0.07 12.00 0.74 38.99 0.66 RG-115597 9.83 1.26RG-115408 >50 0 >50 0.01 RG-115653 19.77 0.53 RG-115665 >50 0 5.49 0.0112.20 0.09 RG-115783 ~15 1.42 3.05 0.93 RG-115866 0.010 0.999

Example 4 Biological (In Vivo) Testing of Compounds

Applicants' ligands are useful in various applications including genetherapy, expression of proteins of interest in host cells, production oftransgenic organisms, and cell-based assays. In vivo induction of areporter enzyme with various ligands of the present invention wasevaluated in a C57BU6 mouse model system containing a gene switch.

Gene Expression Cassettes

The wild-type D, E, and F domains from spruce budworm Choristoneurafumiferana EcR (“CfEcR-DEF”; SEQ ID NO: 1) were mutated [V107 (gtt)→1107(att) and Y127 (tac)→E127 (gag)] and fused to a GAL4 DNA binding domain(“Gal4 DBD1-147”; SEQ ID NO: 2). Helices 1 through 8 of the EF domainsfrom Homo sapiens RXRβ (“HsRXRβ-EF”; nucleotides 1-465 of SEQ ID NO: 4)and helices 9 through 12 of the EF domains of Locusta migratoriaUltraspiracle Protein (“LmUSP-EF”; nucleotides 403-630 of SEQ ID NO: 5)were fused to the transactivation domain from VP16 (“VP16AD”; SEQ ID NO:6), which regulates a reporter gene human secreted alkaline phosphatase(“SEAP”, SEQ ID NO: 12) that was placed under the control of a 6xGAL4response element (SEQ ID NO: 13) and a transthyretin promoter (SEQ IDNO: 14). Each element of the gene switch was on a separate plasmid.Receptor expression was under the control of a CMV promoter (SEQ ID NO:15). Induction was evaluated by the amount of reporter protein expressedin the presence of ligand.

Electroporation of Gene Switch

SEAP expression in serum of mice was evaluated after electroporation ofthe gene switch into mouse quadriceps. Mice were anesthetized with 2μL/g of a mixture of ketamine (100 mg/mL) and xylazine (20 mg/mL).Animals were then shaved, DNA vectors injected into the muscle in avolume of 2×50 μL polyglutamic acid (12 mg/mL water), electrodeconductivity gel applied, and an electrode caliber (1 cm×1 cm; model384) was placed on hind leg. The muscle was electroporated with 200V/cm, 8 times, for 20 msec/pulse, at 1 sec time intervals. Thetransverse electrical field direction was reversed after the animalsreceived half of the pulses. Electroporation was performed with an ECM830 electroporator from BTX Molecular Delivery Systems.

Treatment with Ligand

In some experiments mice received an intraperitoneal injection (IP) of2.6 μmol of ligand in 50 μL of DMSO 3 days after electroporation of thegene switch. In other experiments the concentration of ligand wasdecrease to 26 nmol/50 μL of DMSO/mouse. SEAP expression was evaluated2-11 days after ligand administration. In other experiments ligand wasadministered in rodent chow. The chow was prepared by dissolving 2 g ofligand in 20 mL of acetone and adding it to 1 kg of LabDiet 5010autoclavable chow from Purina Mills. This was thoroughly mixed in aHobart mixer and then mixed for an additional 15 min in a Cross Blendmixer. Animals received chow ad libitum for 1, 2, or 3 days. All valuesare the average from four animals. Background SEAP in sera from animalstreated with vector alone without ligand addition was 0-11 ng/mL serum.

Reporter Assay

Mouse serum was obtained by centrifugation of blood acquired byretroorbital bleeding with a small glass capillary tube. SEAPquantification was determined using a Clontech Great Escapechemiluminescence kit and by comparison with the Clontech SEAP standard.

Table 9: In vivo evaluation of ligand-mediated induction of a mutatedecdysone receptor-based gene switch. SEAP expression in serum of micewas evaluated after electroporation of the gene expression cassettesinto mouse quadriceps. Mice received an IP injection of 2.6 μmol ofligand 3 days after electroporation. SEAP expression was evaluated 2-11days after ligand administration. Each dose group was composed of fouranimals. Percentage of GS™-E ligand induction was determined byaveraging SEAP expression from four animals divided by the average SEAPexpression induced with GS™-E ligand and then multiplying by 100.

Table 10: Induction of gene switch expression with low concentrations ofligand. SEAP expression in serum of mice was evaluated afterelectroporation of the gene expression cassettes into mouse quadriceps.Mice received an IP injection of 26 nmol of ligand or 130 nmol GS™-Eligand 3 days after electroporation. SEAP expression was evaluated 2-7days after ligand administration. Values are the average from 4 animals.

Table 11: Induction of SEAP in C57BL/6 mice with GS™-E ligand orRG-103309 administered in rodent chow. SEAP expression in serum of micewas evaluated after electroporation of the gene expression cassettesinto mouse quadriceps. Mice received GS™-E ligand or RG-103309 in theirfeed (2 g/kg) 3 days after electroporation. Feed was administered adlibitum for 1, 2, or 3 days. After each interval ligand-treated feed wasremoved and animals received untreated feed. Values are the average from4 animals.

TABLE 9 In vivo evaluation of ligand-mediated induction of a mutatedecdysone receptor-based gene switch. Secreted Alkaline Phosphatase(percentage of GS ™-E ligand induction) Compound. Day 2 Day 3 Day 11RG-101382 92 81 890 RG-102317 112 116 61 RG-101523 85 79 1,116 RG-101494136 138 ND RG-115613 2 1 ND RG-115575 74 78 69 RG-115637 12 7 0RG-115517 4 3 ND RG-115092 8 2 ND RG-115009 4 3 ND GS ™-E ligand 100 100100 RG-103309 251 298 ND RG-103451 76 73 371 RG-115819 172 215 3,008RG-115820 82 63 399 RG-115823 129 183 2,652 RG-115824 102 101 415RG-115832 147 189 4,183 RG-115831 118 121 105 RG-115830 120 158 3,558RG-115829 72 83 687 ND—not determined.

TABLE 10 Induction of gene switch expression with low concentrations ofligand. Secreted Alkaline Phosphatase (ng/ml mouse sera) Compound Day 2Day 3 Day 7 GS ™-E ligand(130 652 1,780 139 nmol) RG-103309 (26 nmol)3,428 2,800 143 RG-115819 (26 nmol) 5,984 4,096 453 RG-115823 (26 nmol)3,788 2,705 373 RG-115832 (26 nmol) 2,349 1,807 149 RG-115830 (26 nmol)6,835 5,339 590 RG-115856 (26 nmol) 2,292 2,350 ND RG-115857 (26 nmol)574 401 ND RG-115858 (26 nmol) 13,661 11,820 ND RG-115864 (26 nmol)6,722 5,652 ND ND—not determined.

TABLE 11 Induction of gene switch expression with ligands administeredin rodent’s feed. Secreted Alkaline Phosphatase (ng/ml mouse sera)¹Compound (dose period) Day 2 Day 3 Day 7 GS ™-E ligand (1 day) 7,3027,670 3,784 GS ™-E ligand (2 day) 13,046 15,831 8,816 GS ™-E ligand (3day) 8,064 11,392 8,372 RG-103309 (1 day) 9,003 14,850 5,172 RG-103309(2 day) 6,971 16,518 7,460 RG-103309 (3 day) 11,126 20,373 11,549

In addition, one of ordinary skill in the art is also able to predictthat the ligands disclosed herein will also work to modulate geneexpression in various cell types described above using gene expressionsystems based on group H and group B nuclear receptors.

1. A compound of the general formula:

wherein X and X′ are independently O or S; A is unsubstituted orsubstituted phenyl wherein the substituents are independently 1 to 5 H;halo; nitro; cyano; hydroxy; amino (—NR^(a)R^(b)); alkylaminolkyl(—(CH₂)_(n)NR^(a)R^(b)); (C₁-C₆)alkyl; (C₁-C₆)haloalkyl;(C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy; phenoxy;(C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;(C₁-C₆)alkenyloxy(C₁-C₆)alkyl; (C₁-C₆)alkoxy(C₁-C₆)alkoxy;(C₁-C₆)alkanoyloxy(C₁-C₆)alkyl; (C₂-C₆)alkenyl optionally substitutedwith halo, cyano, (C₁-C₄)alkyl, or (C₁-C₄)alkoxy; (C₂-C₆)alkynyloptionally substituted with halo or (C₁-C₄)alkyl; formyl; carboxy;(C₁-C₆)alkylcarbonyl; (C₁-C₆)haloalkylcarbonyl; benzoyl;(C₁-C₆)alkoxycarbonyl; (C₁-C₆)haloalkoxycarbonyl; (C₁-C₆)alkanoyloxy(—OCOR^(a)); carboxamido (—CONR^(a)R^(b)); amido (—NR^(a)COR^(b));alkoxycarbonylamino (—NR^(a)CO₂R^(b)); alkylaminocarbonylamino(—NR^(a)CONR^(b)R^(c)); mercapto; (C₁-C₆)alkylthio;(C₁-C₆)alkylsulfonyl; (C₁-C₆)alkylsulfonyl(C₁-C₆)alkyl;(C₁-C₆)alkylsulfoxido (—S(O)R^(a)); (C₁-C₆)alkylsulfoxido(C₁-C₆)alkyl—(CH₂)_(n)S(O)R^(a)); sulfaraido (—SO₂NR^(a)R^(b)); —SO₃H; orunsubstituted or substituted phenyl wherein the substituents areindependently 1 to 3 halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino;or when one or both of two adjacent positions on the phenyl ring aresubstituted, the attached atoms may form the phenyl-connecting terminiof a linkage selected from the group consisting of (—OCH₂O—),(—OCH(CH₃)O—), (—OCH₂CH₂O—), (—OCH(CH₃)CH₂O—), (—S-CH═N—), (—CH₂OCH₂O—),(—O(CH₂)₃—), (═N—O—N═), (—CH═CH—NH—), (—OCF₂O—), (—NH—CH═N—),(—CH₂CH₂O—), and (—(CH₂)₄—); B is (a) unsubstituted or substitutedphenyl wherein the substituents are independently 1 to 5 H; halo; nitro;cyano; hydroxy; amino (—NR^(a)R^(b)); alkylaminolkyl(—(CH₂)_(n)NR^(a)R^(b)); (C₁-C₆)alkyl; (C₁-C₆)haloalkyl;(C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy; phenoxy;(C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;(C₁-C₆)alkenyloxy(C₁-C₆)alkyl; (C₁-C₆)alkoxy(C₁-C₆)alkoxy;(C₁-C₆)alkanoyloxy(C₁-C₆)alkyl; (C₂-C₆)alkenyl optionally substitutedwith halo, cyano, (C₁-C₄) alkyl, or (C₁-C₄)alkoxy; (C₂-C₆)alkynyloptionally substituted with halo or (C₁-C₄)alkyl; formyl; carboxy;(C₁-C₆)alkylcarbonyl; (C₁-C₆)haloalkylcarbonyl; benzoyl;(C₁-C₆)alkoxycarbonyl; (C₁-C₆)haloalkoxycarbonyl; (C₁-C₆)alkanoyloxy(—OCOR^(a)); carboxamido (—CONR^(a)R^(b)); amido (—NR^(a)COR^(b));alkoxycarbonylamino (—NR^(a)CO₂R^(b)); alkylaminocarbonylamino(—NR^(a)CONR^(b)R^(c)); mercapto; (C₁-C₆)alkylthio; (C₁-C₆)alkylsulfonyl; (C₁-C₆)alkylsulfonyl(C₁-C₆)alkyl; (C₁-C₆)alkylsulfoxido(—SH(O)R^(a)); (C₁-C₆)alkylsulfoxido(C₁-C₆)alkyl (—CH₂)_(n)S(O)R^(a));sulfamido (—SO₂NR^(a)R^(b)); —SO₃H; —CH═N—NHC(O)NR^(a)R^(b);—CH═N—NHC(O)C(O)NR^(a)R^(b); or unsubstituted or substituted phenylwherein the substituents are independently 1 to 3 halo, nitro,(C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or when one or both of twoadjacent positions on the phenyl ring are substituted, the attachedatoms may form the phenyl-connecting termini of a linkage selected fromthe group consisting of (—OCH₂O—), (—OCH(CH₃)O—), (—OCH₂CH₂O—),(—OCH(CH₃)CH₂O—), (—S-CH═N—), (—CH₂OCH₂O—), (—O(CH₂)₃—), (═N—O—N═),(—CH═CH—NH—), (—OCF₂O—), (—NH—CH═N—), (—CH₂CH₂O—), and (—(CH₂)₄—); (b)unsubstituted 6-membered heterocycle or substituted 6-memberedheterocycle having 1-3 nitrogen atoms and 3-5 nuclear carbon atoms wherethe substituents are from one to three of the same or different halo;nitro; hydroxy; (C₁-C₆)alkyl; (C₁-C₆)alkoxy; (C₁-C₆)thioalkoxy; carboxy;(C₁-C₆)alkoxycarbonyl; (C₁-C₆)arboxyalkyl; (C₁-C₆)alkoxycarbonylalkylhaving independently the stated number of carbon atoms in each aikylgroup; —CONR^(a)R^(b); amino; (C₁-C₆)alkylamino; (C₁-C₆)dialkylaminohaving independently the stated number of carbon atoms in each aikylgroup; haloalkyl; —CH═N—NHC(O)NR^(a)R^(b); or—CH═N—NHC(O)C(O)NR^(a)R^(b); or (c) 5-benzimidazolyl;1-trityl-5-benzimidazolyl; 3-trityl-5-benzimidazolyl; 1H-indazole-3-yl;l-trityl-1H-indazole-3-yl; or 1-(C₁-C₆)alkyl-1H-indole-2-yl; E isunsubstituted or substituted (C₄-C₁₀) branched aikyl wherein thesubstituents are independently 1-4 cyano; halo; (C₅-C₆)cycloalkyl;phenyl; (C₂-C₃)alkenyl; hydroxy, (C₁-C₆)alkoxy; carboxy;(C₁-C₆)alkoxycarbonyl; formyl; (C₁-C₆)trialkylsilyloxy havingindependently the stated number of carbon atoms in each aikyl group;—CH═N—OR^(a); —CH═N—R^(d); —CH═N—NHC(O)NR^(a)R^(b); or—CH═N—NHC(O)C(O)NR^(a)R^(b); wherein R^(a), R^(b), and R^(c) areindependently H, (C₁-C₆)alkyl, or phenyl; R^(d) is hydroxy(C₁-C₆)alkyl;and n=1-4; and G is H or CN; provided that: 1) when E is unsubstitutedor substituted (C₄-C₁₀) branched aikyl wherein the substituents areindependently 1-4 cyano; halo; (C₂-C₃)alkenyl; carboxy; or(C₁-C₆)alkoxycarbonyl; then B is: (a) substituted phenyl which bears atleast one —CH═N—NHC(O)NR^(a)R^(b) or —CH═N—NHC(O)C(O)NR^(a)R^(b) group;(b) substituted 6-membered heterocycle having 1-3 nitrogen atoms and 3-5nuclear carbon atoms which bears at least one haloalkyl group; or (c)5-benzimidazolyl; 1-trityl-5-benzimidazolyl; 3-trityl-5-benzimidazolyl;1H-indazole-3-yl; 1-trityl-1H-indazole-3-yl; or1-(C₁-C₆)alkyl-1H-indole-2-yl; wherein R^(a), R^(b) are independently H,(C₁-C₆)alkyl, or phenyl; or 2) when E is a substituted (C₄-C₁₀) branchedalkyi which bears at least one of phenyl; hydroxy, (C₁-C₆)alkoxy; orformyl; then B is: (a) substituted phenyl which bears at least one—CH═N—NHC(O)NR^(a)R^(b) or —CH—N—NHC(O)C(O)NR^(a)R^(b) group; (b)substituted or unsubstituted 6-membered heterocycle having 1-3 nitrogenatoms and 3-5 nuclear carbon atoms; or (c) 5-benzimidazolyl;1-trityl-5-benzimidazolyl; 3-trityl-5-benzimidazolyl; 1H-indazole-3-yl;1-trityl-1H-indazole-3-yl; or 1-(C₁-C₆)alkyl-1H-indole-2-yl; whereinR^(a) and R^(b) are independently H, (C₁—C₆)alkyl, or phenyl.
 2. Thecompound of claim 1, wherein X and X′ are O and G is H. 3.-14.(canceled)
 15. The compound of claim 1, wherein said haloalkyl is —CF₃.